Modified stem cell memory T cells, methods of making and methods of using same

ABSTRACT

The disclosure provides a method of producing modified stem memory T cells (e.g. CAR-T cells) for administration to a subject as, for example an adoptive cell therapy.

RELATED APPLICATIONS

This application is a Continuation Application of InternationalApplication No. PCT/US2017/054799 filed on Oct. 2, 2017, which claimsthe benefit of provisional applications U.S. Ser. No. 62/402,707 filedSep. 30, 2016, U.S. Ser. No. 62/502,508 filed May 5, 2017, U.S. Ser. No.62/553,058 filed Aug. 31, 2017 and U.S. Ser. No. 62/556,309 filed Sep.8, 2017, the contents of each of which are herein incorporated byreference in their entirety.

INCORPORATION OF SEQUENCE LISTING

The contents of the text filed named “POTH-012_C01US_SeqList.txt”, whichwas created on Nov. 13, 2017 and is 111 KB in size, are herebyincorporated by reference in their entirety.

FIELD OF THE DISCLOSURE

The disclosure is directed to molecular biology, and more, specifically,to methods of making and using modified stem-cell memory T cells.

BACKGROUND

There has been a long-felt but unmet need in the art for a method ofproducing modified stem-cell memory T cells for administration to asubject as, for example, an adoptive cell therapy. The disclosureprovides a solution to this long-felt but unmet need.

SUMMARY

Unlike traditional biologics and chemotherapeutics, modified-T cells ofthe disclosure possess the capacity to rapidly reproduce upon antigenrecognition, thereby potentially obviating the need for repeattreatments. To achieve this, modified-T cells of the disclosure must notonly drive tumor destruction initially, but must also persist in thepatient as a stable population of viable memory T cells to preventpotential cancer relapses. Thus, intensive efforts have been focused onthe development of antigen receptor molecules that do not cause T cellexhaustion through antigen-independent (tonic) signaling, as well as ofa modified-T cell product containing early memory cells, especially stemcell memory (T_(SCM)). Stem cell-like modified-T cells of the disclosureexhibit the greatest capacity for self-renewal and multipotent capacityto derive central memory (T_(CM)), effector memory (T_(EM)) and effectorT cells (T_(E)), thereby producing better tumor eradication andlong-term modified-T cell engraftment. Modified-T cells of thedisclosure include, but are not limited to, those cells that express anantigen receptor comprising a protein scaffold of the disclosure.Modified-T cells of the disclosure include, but are not limited to,those cells that express a chimeric antigen receptor (CAR) (i.e. CAR-Tcells of the disclosure). Chimeric antigen receptors (CARs) of thedisclosure may comprise one or more sequences that each specificallybind an antigen, including, but not limited to, a single chain antibody(e.g. a scFv), a sequence comprising one or more fragments of anantibody (e.g. a VHH, referred to in the context of a CAR as a VCAR), anantibody mimic, and a Centyrin (referred to in the context of a CAR as aCARTyrin).

Modified cells of the disclosure may be further subjected to genomicediting. For example, a genomic editing construct may be introduced intothe modified cells of the disclosure in a transposon or other means ofdelivery through electroporation or nucleofection and allowed tointegrate into the genome of the cell during the following incubationphase. The resultant cell is a modified T cell with an edited genomethat retains a stem-like phenotype. This modified T cell with an editedgenome that retains a stem-like phenotype may be used as a cellulartherapy. Alternatively, or in addition, modified cells of the disclosuremay be subject to a first electroporation or nucleofection and asubsequent electroporation or nucleofection to introduce a genomicediting construct.

Specifically, the disclosure provides a method of producing a modifiedstem memory T cell (T_(SCM)), comprising introducing into a primaryhuman T cell (a) a transposon composition comprising a transposoncomprising an antigen receptor or a therapeutic protein and (b) atransposase composition comprising a transposase or a sequence encodingthe transposase; to produce a modified T cell, wherein the modified Tcell expresses one or more cell-surface marker(s) of a stem memory Tcell (T_(SCM)), thereby producing a modified stem memory T cell(T_(SCM)). The disclosure provides a method of producing a plurality ofmodified stem memory T cells (T_(SCM)), comprising introducing into aplurality of primary human T cell (a) a transposon compositioncomprising a transposon comprising an antigen receptor or a therapeuticprotein and (b) a transposase composition comprising a transposase or asequence encoding the transposase; to produce a plurality of modified Tcells, wherein at least 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%,50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or any percentage inbetween of the plurality of modified T cells expresses one or morecell-surface marker(s) of a stem memory T cell (T_(SCM)), therebyproducing a plurality of modified stem memory T cells (T_(SCM)). Incertain embodiments, the method produces a plurality of modified Tcells, wherein at least 25% of the plurality of modified T cellsexpresses one or more cell-surface marker(s) of a stem memory T cell(T_(SCM)), thereby producing a plurality of modified stem memory T cells(T_(SCM)). In certain embodiments, the method produces a plurality ofmodified T cells, wherein at least 50% of the plurality of modified Tcells expresses one or more cell-surface marker(s) of a stem memory Tcell (T_(SCM)), thereby producing a plurality of modified stem memory Tcells (T_(SCM)). In certain embodiments, the method produces a pluralityof modified T cells, wherein at least 60% of the plurality of modified Tcells expresses one or more cell-surface marker(s) of a stem memory Tcell (T_(SCM)), thereby producing a plurality of modified stem memory Tcells (T_(SCM)). In certain embodiments, the method produces a pluralityof modified T cells, wherein at least 75% of the plurality of modified Tcells expresses one or more cell-surface marker(s) of a stem memory Tcell (T_(SCM)), thereby producing a plurality of modified stem memory Tcells (T_(SCM)). In certain embodiments, the method produces a pluralityof modified T cells, wherein at least 80% of the plurality of modified Tcells expresses one or more cell-surface marker(s) of a stem memory Tcell (T_(SCM)), thereby producing a plurality of modified stem memory Tcells (T_(SCM)). In certain embodiments, the method produces a pluralityof modified T cells, wherein at least 85% of the plurality of modified Tcells expresses one or more cell-surface marker(s) of a stem memory Tcell (T_(SCM)), thereby producing a plurality of modified stem memory Tcells (T_(SCM)). In certain embodiments, the method produces a pluralityof modified T cells, wherein at least 90% of the plurality of modified Tcells expresses one or more cell-surface marker(s) of a stem memory Tcell (T_(SCM)), thereby producing a plurality of modified stem memory Tcells (T_(SCM)). In certain embodiments, the method produces a pluralityof modified T cells, wherein at least 95% of the plurality of modified Tcells expresses one or more cell-surface marker(s) of a stem memory Tcell (T_(SCM)), thereby producing a plurality of modified stem memory Tcells (T_(SCM)). In certain embodiments, the cell-surface markerscomprise CD62L and CD45RA. In certain embodiments, the cell-surfacemarkers of the CAR-T_(SCM) comprise one or more of CD62L, CD45RA, CD28,CCR7, CD127, CD45RO, CD95, CD95 and IL-2Rβ. In certain embodiments, thecell-surface markers of the CAR-T_(SCM) comprise one or more of CD45RA,CD95, IL-2Rβ, CR7, and CD62L. In certain embodiments of this method, thetransposon is a plasmid DNA transposon with a sequence encoding theantigen receptor or the therapeutic protein is flanked by twocis-regulatory insulator elements. In certain embodiments, thetransposon is a piggyBac transposon. In certain embodiments, and, inparticular, those embodiments wherein the transposon is a piggyBactransposon, the transposase is a piggyBac™ or a Super piggyBac™ (SPB)transposase.

In certain embodiments of the methods of the disclosure, the transposonis a plasmid DNA transposon with a sequence encoding the antigenreceptor or the therapeutic protein is flanked by two cis-regulatoryinsulator elements. In certain embodiments, the transposon is a piggyBactransposon. In certain embodiments, and, in particular, thoseembodiments wherein the transposon is a piggyBac transposon, thetransposase is a piggyBac™ or a Super piggyBac™ (SPB) transposase. Incertain embodiments, and, in particular, those embodiments wherein thetransposase is a Super piggyBac™ (SPB) transposase, the sequenceencoding the transposase is an mRNA sequence.

In certain embodiments of the methods of the disclosure, the transposaseenzyme is a piggyBac™ (PB) transposase enzyme. The piggyBac (PB)transposase enzyme may comprise or consist of an amino acid sequence atleast 75%, 80%, 85%, 90%, 95%, 99% or any percentage in betweenidentical to:

(SEQ ID NO: 4)  1 MGSSLDDEHI LSALLQSDDE LVGEDSDSEI SDHVSEDDVQ SDTEEAFIDE VHEVQPTSSG 61 SEILDEQNVI EQPGSSLASN RILTLPQRTI RGKNKHCWST SKSTRRSRVS ALNIVRSQRG121 PTRMCRNIYD PLLCFKLFFT DEIISEIVKW TNAEISLKRR ESMTGATFRD TNEDEIYAFF181 GILVMTAVRK DNHMSTDDLF DRSLSMVYVS VMSRDRFDFL IRCLRMDDKS IRPTLRENDV241 FTPVRKIWDL FIHQCIQNYT PGAHLTIDEQ LLGFRGRCPF RMYIPNKPSK YGIKILMMCD301 SGYKYMINGM PYLGRGTQTN GVPLGEYYVK ELSKPVHGSC RNITCDNWFT SIPLAKNLLQ361 EPYKLTIVGT VRSNKREIPE VLKNSRSRPV GTSMFCFDGP LTLVSYKPKP AKMVYLLSSC421 DEDASINEST GKPQMVMYYN QTKGGVDTLD QMCSVMTCSR KTNRWPMALL YGMINIACIN481 SFIIYSHNVS SKGEKVQSRK KFMRNLYMSL TSSFMRKRLE APTLKRYLRD NISNILPNEV541 PGTSDDSTEE PVMKKRTYCT YCPSKIRRKA NASCKKCKKV ICREHNIDMC QSCF.

In certain embodiments of the methods of the disclosure, the transposaseenzyme is a piggyBac™ (PB) transposase enzyme that comprises or consistsof an amino acid sequence having an amino acid substitution at one ormore of positions 30, 165, 282, or 538 of the sequence:

(SEQ ID NO: 4)  1 MGSSLDDEHI LSALLQSDDE LVGEDSDSEI SDHVSEDDVQ SDTEEAFIDE VHEVQPTSSG 61 SEILDEQNVI EQPGSSLASN RILTLPQRTI RGKNKHCWST SKSTRRSRVS ALNIVRSQRG121 PTRMCRNIYD PLLCFKLFFT DEIISEIVKW TNAEISLKRR ESMTGATFRD TNEDEIYAFF181 GILVMTAVRK DNHMSTDDLF DRSLSMVYVS VMSRDRFDFL IRCLRMDDKS IRPTLRENDV241 FTPVRKIWDL FIHQCIQNYT PGAHLTIDEQ LLGFRGRCPF RMYIPNKPSK YGIKILMMCD301 SGYKYMINGM PYLGRGTQTN GVPLGEYYVK ELSKPVHGSC RNITCDNWFT SIPLAKNLLQ361 EPYKLTIVGT VRSNKREIPE VLKNSRSRPV GTSMFCFDGP LTLVSYKPKP AKMVYLLSSC421 DEDASINEST GKPQMVMYYN QTKGGVDTLD QMCSVMTCSR KTNRWPMALL YGMINIACIN481 SFIIYSHNVS SKGEKVQSRK KFMRNLYMSL TSSFMRKRLE APTLKRYLRD NISNILPNEV541 PGTSDDSTEE PVMKKRTYCT YCPSKIRRKA NASCKKCKKV ICREHNIDMC QSCF.

In certain embodiments, the transposase enzyme is a piggyBac™ (PB)transposase enzyme that comprises or consists of an amino acid sequencehaving an amino acid substitution at two or more of positions 30, 165,282, or 538 of the sequence of SEQ ID NO: 4. In certain embodiments, thetransposase enzyme is a piggyBac™ (PB) transposase enzyme that comprisesor consists of an amino acid sequence having an amino acid substitutionat three or more of positions 30, 165, 282, or 538 of the sequence ofSEQ ID NO: 4. In certain embodiments, the transposase enzyme is apiggyBac™ (PB) transposase enzyme that comprises or consists of an aminoacid sequence having an amino acid substitution at each of the followingpositions 30, 165, 282, and 538 of the sequence of SEQ ID NO: 4. Incertain embodiments, the amino acid substitution at position 30 of thesequence of SEQ ID NO: 4 is a substitution of a valine (V) for anisoleucine (I). In certain embodiments, the amino acid substitution atposition 165 of the sequence of SEQ ID NO: 4 is a substitution of aserine (S) for a glycine (G). In certain embodiments, the amino acidsubstitution at position 282 of the sequence of SEQ ID NO: 4 is asubstitution of a valine (V) for a methionine (M). In certainembodiments, the amino acid substitution at position 538 of the sequenceof SEQ ID NO: 4 is a substitution of a lysine (K) for an asparagine (N).

In certain embodiments of the methods of the disclosure, the transposaseenzyme is a Super piggyBac™ (SPB) transposase enzyme. In certainembodiments, the Super piggyBac™ (SPB) transposase enzymes of thedisclosure may comprise or consist of the amino acid sequence of thesequence of SEQ ID NO: 4 wherein the amino acid substitution at position30 is a substitution of a valine (V) for an isoleucine (I), the aminoacid substitution at position 165 is a substitution of a serine (S) fora glycine (G), the amino acid substitution at position 282 is asubstitution of a valine (V) for a methionine (M), and the amino acidsubstitution at position 538 is a substitution of a lysine (K) for anasparagine (N). In certain embodiments, the Super piggyBac™ (SPB)transposase enzyme may comprise or consist of an amino acid sequence atleast 75%, 80%, 85%, 90%, 95%, 99% or any percentage in betweenidentical to:

(SEQ ID NO: 5)  1 MGSSLDDEHI LSALLQSDDE LVGEDSDSEV SDHVSEDDVQ SDTEEAFIDE VHEVQPTSSG 61 SEILDEQNVI EQPGSSLASN RILTLPQRTI RGKNKHCWST SKSTRRSRVS ALNIVRSQRG121 PTRMCRNIYD PLLCFKLFFT DEIISEIVKW TNAEISLKRR ESMTSATFRD TNEDEIYAFF181 GILVMTAVRK DNHMSTDDLF DRSLSMVYVS VMSRDRFDFL IRCLRMDDKS IRPTLRENDV241 FTPVRKIWDL FIHQCIQNYT PGAHLTIDEQ LLGFRGRCPF RVYIPNKPSK YGIKILMMCD301 SGTKYMINGM PYLGRGTQTN GVPLGEYYVK ELSKPVHGSC RNITCDNWFT SIPLAKNLLQ361 EPYKLTIVGT VRSNKREIPE VLKNSRSRPV GTSMFCFDGP LTLVSYKPKP AKMVYLLSSC421 DEDASINEST GKPQMVMYYN QTKGGVDTLD QMCSVMTCSR KTNRWPMALL YGMINIACIN481 SFIIYSHNVS SKGEKVQSRK KFMRNLYMSL TSSFMRKRLE APTLKRYLRD NISNILPKEV541 PGTSDDSTEE PVMKKRTYCT YCPSKIRRKA NASCKKCKKV ICREHNIDMC QSCF.

In certain embodiments of the methods of the disclosure, including thoseembodiments wherein the transposase comprises the above-describedmutations at positions 30, 165, 282 and/or 538, the piggyBac™ or SuperpiggyBac™ transposase enzyme may further comprise an amino acidsubstitution at one or more of positions 3, 46, 82, 103, 119, 125, 177,180, 185, 187, 200, 207, 209, 226, 235, 240, 241, 243, 258, 296, 298,311, 315, 319, 327, 328, 340, 421, 436, 456, 470, 486, 503, 552, 570 and591 of the sequence of SEQ ID NO: 4 or SEQ ID NO: 5. In certainembodiments, including those embodiments wherein the transposasecomprises the above-described mutations at positions 30, 165, 282 and/or538, the piggyBac™ or Super piggyBac™ transposase enzyme may furthercomprise an amino acid substitution at one or more of positions 46, 119,125, 177, 180, 185, 187, 200, 207, 209, 226, 235, 240, 241, 243, 296,298, 311, 315, 319, 327, 328, 340, 421, 436, 456, 470, 485, 503, 552 and570. In certain embodiments, the amino acid substitution at position 3of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of an asparagine (N)for a serine (S). In certain embodiments, the amino acid substitution atposition 46 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of aserine (S) for an alanine (A). In certain embodiments, the amino acidsubstitution at position 46 of SEQ ID NO: 4 or SEQ ID NO: 5 is asubstitution of a threonine (T) for an alanine (A). In certainembodiments, the amino acid substitution at position 82 of SEQ ID NO: 4or SEQ ID NO: 5 is a substitution of a tryptophan (W) for an isoleucine(I). In certain embodiments, the amino acid substitution at position 103of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a proline (P) for aserine (S). In certain embodiments, the amino acid substitution atposition 119 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of aproline (P) for an arginine (R). In certain embodiments, the amino acidsubstitution at position 125 of SEQ ID NO: 4 or SEQ ID NO: 5 is asubstitution of an alanine (A) a cysteine (C). In certain embodiments,the amino acid substitution at position 125 of SEQ ID NO: 4 or SEQ IDNO: 5 is a substitution of a leucine (L) for a cysteine (C). In certainembodiments, the amino acid substitution at position 177 of SEQ ID NO: 4or SEQ ID NO: 5 is a substitution of a lysine (K) for a tyrosine (Y). Incertain embodiments, the amino acid substitution at position 177 of SEQID NO: 4 or SEQ ID NO: 5 is a substitution of a histidine (H) for atyrosine (Y). In certain embodiments, the amino acid substitution atposition 180 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of aleucine (L) for a phenylalanine (F). In certain embodiments, the aminoacid substitution at position 180 of SEQ ID NO: 4 or SEQ ID NO: 5 is asubstitution of an isoleucine (I) for a phenylalanine (F). In certainembodiments, the amino acid substitution at position 180 of SEQ ID NO: 4or SEQ ID NO: 5 is a substitution of a valine (V) for a phenylalanine(F). In certain embodiments, the amino acid substitution at position 185of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a leucine (L) for amethionine (M). In certain embodiments, the amino acid substitution atposition 187 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of aglycine (G) for an alanine (A). In certain embodiments, the amino acidsubstitution at position 200 of SEQ ID NO: 4 or SEQ ID NO: 5 is asubstitution of a tryptophan (W) for a phenylalanine (F). In certainembodiments, the amino acid substitution at position 207 of SEQ ID NO: 4or SEQ ID NO: 5 is a substitution of a proline (P) for a valine (V). Incertain embodiments, the amino acid substitution at position 209 of SEQID NO: 4 or SEQ ID NO: 5 is a substitution of a phenylalanine (F) for avaline (V). In certain embodiments, the amino acid substitution atposition 226 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of aphenylalanine (F) for a methionine (M). In certain embodiments, theamino acid substitution at position 235 of SEQ ID NO: 4 or SEQ ID NO: 5is a substitution of an arginine (R) for a leucine (L). In certainembodiments, the amino acid substitution at position 240 of SEQ ID NO: 4or SEQ ID NO: 5 is a substitution of a lysine (K) for a valine (V). Incertain embodiments, the amino acid substitution at position 241 of SEQID NO: 4 or SEQ ID NO: 5 is a substitution of a leucine (L) for aphenylalanine (F). In certain embodiments, the amino acid substitutionat position 243 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of alysine (K) for a proline (P). In certain embodiments, the amino acidsubstitution at position 258 of SEQ ID NO: 4 or SEQ ID NO: 5 is asubstitution of a serine (S) for an asparagine (N). In certainembodiments, the amino acid substitution at position 296 of SEQ ID NO: 4or SEQ ID NO: 5 is a substitution of a tryptophan (W) for a leucine (L).In certain embodiments, the amino acid substitution at position 296 ofSEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a tyrosine (Y) for aleucine (L). In certain embodiments, the amino acid substitution atposition 296 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of aphenylalanine (F) for a leucine (L). In certain embodiments, the aminoacid substitution at position 298 of SEQ ID NO: 4 or SEQ ID NO: 5 is asubstitution of a leucine (L) for a methionine (M). In certainembodiments, the amino acid substitution at position 298 of SEQ ID NO: 4or SEQ ID NO: 5 is a substitution of an alanine (A) for a methionine(M). In certain embodiments, the amino acid substitution at position 298of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a valine (V) for amethionine (M). In certain embodiments, the amino acid substitution atposition 311 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of anisoleucine (I) for a proline (P). In certain embodiments, the amino acidsubstitution at position 311 of SEQ ID NO: 4 or SEQ ID NO: 5 is asubstitution of a valine for a proline (P). In certain embodiments, theamino acid substitution at position 315 of SEQ ID NO: 4 or SEQ ID NO: 5is a substitution of a lysine (K) for an arginine (R). In certainembodiments, the amino acid substitution at position 319 of SEQ ID NO: 4or SEQ ID NO: 5 is a substitution of a glycine (G) for a threonine (T).In certain embodiments, the amino acid substitution at position 327 ofSEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of an arginine (R) for atyrosine (Y). In certain embodiments, the amino acid substitution atposition 328 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of avaline (V) for a tyrosine (Y). In certain embodiments, the amino acidsubstitution at position 340 of SEQ ID NO: 4 or SEQ ID NO: 5 is asubstitution of a glycine (G) for a cysteine (C). In certainembodiments, the amino acid substitution at position 340 of SEQ ID NO: 4or SEQ ID NO: 5 is a substitution of a leucine (L) for a cysteine (C).In certain embodiments, the amino acid substitution at position 421 ofSEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a histidine (H) forthe aspartic acid (D). In certain embodiments, the amino acidsubstitution at position 436 of SEQ ID NO: 4 or SEQ ID NO: 5 is asubstitution of an isoleucine (I) for a valine (V). In certainembodiments, the amino acid substitution at position 456 of SEQ ID NO: 4or SEQ ID NO: 5 is a substitution of a tyrosine (Y) for a methionine(M). In certain embodiments, the amino acid substitution at position 470of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a phenylalanine (F)for a leucine (L). In certain embodiments, the amino acid substitutionat position 485 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of alysine (K) for a serine (S). In certain embodiments, the amino acidsubstitution at position 503 of SEQ ID NO: 4 or SEQ ID NO: 5 is asubstitution of a leucine (L) for a methionine (M). In certainembodiments, the amino acid substitution at position 503 of SEQ ID NO: 4or SEQ ID NO: 5 is a substitution of an isoleucine (I) for a methionine(M). In certain embodiments, the amino acid substitution at position 552of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a lysine (K) for avaline (V). In certain embodiments, the amino acid substitution atposition 570 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of athreonine (T) for an alanine (A). In certain embodiments, the amino acidsubstitution at position 591 of SEQ ID NO: 4 or SEQ ID NO: 5 is asubstitution of a proline (P) for a glutamine (Q). In certainembodiments, the amino acid substitution at position 591 of SEQ ID NO: 4or SEQ ID NO: 5 is a substitution of an arginine (R) for a glutamine(Q).

In certain embodiments of the methods of the disclosure, including thoseembodiments wherein the transposase comprises the above-describedmutations at positions 30, 165, 282 and/or 538, the piggyBac™transposase enzyme may comprise or the Super piggyBac™ transposaseenzyme may further comprise an amino acid substitution at one or more ofpositions 103, 194, 372, 375, 450, 509 and 570 of the sequence of SEQ IDNO: 4 or SEQ ID NO: 5. In certain embodiments of the methods of thedisclosure, including those embodiments wherein the transposasecomprises the above-described mutations at positions 30, 165, 282 and/or538, the piggyBac™ transposase enzyme may comprise or the SuperpiggyBac™ transposase enzyme may further comprise an amino acidsubstitution at two, three, four, five, six or more of positions 103,194, 372, 375, 450, 509 and 570 of the sequence of SEQ ID NO: 4 or SEQID NO: 5. In certain embodiments, including those embodiments whereinthe transposase comprises the above-described mutations at positions 30,165, 282 and/or 538, the piggyBac™ transposase enzyme may comprise orthe Super piggyBac™ transposase enzyme may further comprise an aminoacid substitution at positions 103, 194, 372, 375, 450, 509 and 570 ofthe sequence of SEQ ID NO: 4 or SEQ ID NO: 5. In certain embodiments,the amino acid substitution at position 103 of SEQ ID NO: 4 or SEQ IDNO: 5 is a substitution of a proline (P) for a serine (S). In certainembodiments, the amino acid substitution at position 194 of SEQ ID NO: 4or SEQ ID NO: 5 is a substitution of a valine (V) for a methionine (M).In certain embodiments, the amino acid substitution at position 372 ofSEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of an alanine (A) for anarginine (R). In certain embodiments, the amino acid substitution atposition 375 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of analanine (A) for a lysine (K). In certain embodiments, the amino acidsubstitution at position 450 of SEQ ID NO: 4 or SEQ ID NO: 5 is asubstitution of an asparagine (N) for an aspartic acid (D). In certainembodiments, the amino acid substitution at position 509 of SEQ ID NO: 4or SEQ ID NO: 5 is a substitution of a glycine (G) for a serine (S). Incertain embodiments, the amino acid substitution at position 570 of SEQID NO: 4 or SEQ ID NO: 5 is a substitution of a serine (S) for anasparagine (N). In certain embodiments, the piggyBac™ transposase enzymemay comprise a substitution of a valine (V) for a methionine (M) atposition 194 of SEQ ID NO: 4. In certain embodiments, including thoseembodiments wherein the piggyBac™ transposase enzyme may comprise asubstitution of a valine (V) for a methionine (M) at position 194 of SEQID NO: 4, the piggyBac™ transposase enzyme may further comprise an aminoacid substitution at positions 372, 375 and 450 of the sequence of SEQID NO: 4 or SEQ ID NO: 5. In certain embodiments, the piggyBac™transposase enzyme may comprise a substitution of a valine (V) for amethionine (M) at position 194 of SEQ ID NO: 4, a substitution of analanine (A) for an arginine (R) at position 372 of SEQ ID NO: 4, and asubstitution of an alanine (A) for a lysine (K) at position 375 of SEQID NO: 4. In certain embodiments, the piggyBac™ transposase enzyme maycomprise a substitution of a valine (V) for a methionine (M) at position194 of SEQ ID NO: 4, a substitution of an alanine (A) for an arginine(R) at position 372 of SEQ ID NO: 4, a substitution of an alanine (A)for a lysine (K) at position 375 of SEQ ID NO: 4 and a substitution ofan asparagine (N) for an aspartic acid (D) at position 450 of SEQ ID NO:4.

The disclosure provides a method of producing a modified stem memory Tcell (T_(SCM)), comprising introducing into a primary human T cell (a) atransposon composition comprising a transposon comprising an antigenreceptor or a therapeutic protein and (b) a transposase compositioncomprising a transposase or a sequence encoding the transposase; toproduce a modified T cell, wherein the modified T cell expresses one ormore cell-surface marker(s) of a stem memory T cell (T_(SCM)), therebyproducing a modified stem memory T cell (T_(SCM)). The disclosureprovides a method of producing a plurality of modified stem memory Tcells (T_(SCM)), comprising introducing into a plurality of primaryhuman T cell (a) a transposon composition comprising a transposoncomprising an antigen receptor or a therapeutic protein and (b) atransposase composition comprising a transposase or a sequence encodingthe transposase; to produce a plurality of modified T cells, wherein atleast 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 65%,70%, 75%, 80%, 85%, 90%, 95%, 99% or any percentage in between of theplurality of modified T cells expresses one or more cell-surfacemarker(s) of a stem memory T cell (T_(SCM)), thereby producing aplurality of modified stem memory T cells (T_(SCM)). In certainembodiments, the method produces a plurality of modified T cells,wherein at least 25% of the plurality of modified T cells expresses oneor more cell-surface marker(s) of a stem memory T cell (T_(SCM)),thereby producing a plurality of modified stem memory T cells (T_(SCM)).In certain embodiments, the method produces a plurality of modified Tcells, wherein at least 50% of the plurality of modified T cellsexpresses one or more cell-surface marker(s) of a stem memory T cell(T_(SCM)), thereby producing a plurality of modified stem memory T cells(T_(SCM)). In certain embodiments, the method produces a plurality ofmodified T cells, wherein at least 60% of the plurality of modified Tcells expresses one or more cell-surface marker(s) of a stem memory Tcell (T_(SCM)), thereby producing a plurality of modified stem memory Tcells (T_(SCM)). In certain embodiments, the method produces a pluralityof modified T cells, wherein at least 75% of the plurality of modified Tcells expresses one or more cell-surface marker(s) of a stem memory Tcell (T_(SCM)), thereby producing a plurality of modified stem memory Tcells (T_(SCM)). In certain embodiments, the method produces a pluralityof modified T cells, wherein at least 80% of the plurality of modified Tcells expresses one or more cell-surface marker(s) of a stem memory Tcell (T_(SCM)), thereby producing a plurality of modified stem memory Tcells (T_(SCM)). In certain embodiments, the method produces a pluralityof modified T cells, wherein at least 85% of the plurality of modified Tcells expresses one or more cell-surface marker(s) of a stem memory Tcell (T_(SCM)), thereby producing a plurality of modified stem memory Tcells (T_(SCM)). In certain embodiments, the method produces a pluralityof modified T cells, wherein at least 90% of the plurality of modified Tcells expresses one or more cell-surface marker(s) of a stem memory Tcell (T_(SCM)), thereby producing a plurality of modified stem memory Tcells (T_(SCM)). In certain embodiments, the method produces a pluralityof modified T cells, wherein at least 95% of the plurality of modified Tcells expresses one or more cell-surface marker(s) of a stem memory Tcell (T_(SCM)), thereby producing a plurality of modified stem memory Tcells (T_(SCM)). In certain embodiments, the cell-surface markerscomprise CD62L and CD45RA. In certain embodiments, the cell-surfacemarkers of the CAR-T_(SCM) comprise one or more of CD62L, CD45RA, CD28,CCR7, CD127, CD45RO, CD95, CD95 and IL-2Rβ. In certain embodiments, thecell-surface markers of the CAR-T_(SCM) comprise one or more of CD45RA,CD95, IL-2Rβ, CR7, and CD62L. In certain embodiments of this method, thetransposon is a Sleeping Beauty transposon. In certain embodiments, and,in particular, those embodiments wherein the transposon is a SleepingBeauty transposon, the transposase is a Sleeping Beauty transposase or ahyperactive Sleeping Beauty transposase (SB100X).

In certain embodiments of the methods of the disclosure, the SleepingBeauty transposase enzyme comprises an amino acid sequence at least 75%,80%, 85%, 90%, 95%, 99% or any percentage in between identical to:

(SEQ ID NO: 6)  1 MGKSKEISQD LRKKIVDLHK SGSSLGAISK RLKVPRSSVQ TIVRKYKHHG TTQPSYRSGR 61 RRYLSPRDER TLVRKVQINP RTTAKDLVKM LEETGTKVSI STVKRVLYRH NLKGRSARKK121 PLLQNRHKKA RLRFATAHGD KDRTFWRNVL WSDETKIELF GHNDHRYVWR KKGEACKPKN181 TIPTVKHGGG SIMLWGCFAA GGTGALHKID GIMRKENYVD ILKQHLKTSV RKLKLGRKWV241 FQMDNDPKHT SKVVAKWLKD NKVKVLEWPS QSPDLNPIEN LWAELKKRVR ARRPTNLTQL301 HQLCQEEWAK IHPTYCGKLV EGYPKRLTQV KQFKGNATKY.

In certain embodiments of the methods of the disclosure, the hyperactiveSleeping Beauty (SB100X) transposase enzyme comprises an amino acidsequence at least 75%, 80%, 85%, 90%, 95%, 99% or any percentage inbetween identical to:

(SEQ ID NO: 7)  1 MGKSKEISQD LRKRIVDLHK SGSSLGAISK RLAVPRSSVQ TIVRKYKHHG TTQPSYRSGR 61 RRYLSPRDER TLVRKVQINP RTTAKDLVKM LEETGTKVSI STVKRVLYRH NLKGHSARKK121 PLLQNRHKKA RLRFATAHGD KDRTFWRNVL WSDETKIELF GHNDHRYVWR KKGEACKPKN181 TIPTVKHGGG SIMLWGCFAA GGTGALHKID GIMDAVQYVD ILKQHLKTSV RKLKLGRKWV241 FQHDNDPKHT SKVVAKWLKD NKVKVLEWPS QSPDLNPIEN LWAELKKRVR ARRPTNLTQL301 HQLCQEEWAK IHPNYCGKLV EGYPKRLTQV KQFKGNATKY.

The disclosure provides a method of producing a modified stem memory Tcell (T_(SCM)), comprising introducing into a primary human T cell (a) atransposon composition comprising a transposon comprising an antigenreceptor or a therapeutic protein and (b) a transposase compositioncomprising a transposase or a sequence encoding the transposase; toproduce a modified T cell, wherein the modified T cell expresses one ormore cell-surface marker(s) of a stem memory T cell (T_(SCM)), therebyproducing a modified stem memory T cell (T_(SCM)). The disclosureprovides a method of producing a plurality of modified stem memory Tcells (T_(SCM)), comprising introducing into a plurality of primaryhuman T cell (a) a transposon composition comprising a transposoncomprising an antigen receptor and (b) a transposase compositioncomprising a transposase or a sequence encoding the transposase; toproduce a plurality of modified T cells, wherein at least 2%, 5%, 10%,15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 95%, 99% or any percentage in between of the plurality of modifiedT cells expresses one or more cell-surface marker(s) of a stem memory Tcell (T_(SCM)), thereby producing a plurality of modified stem memory Tcells (T_(SCM)). In certain embodiments, the method produces a pluralityof modified T cells, wherein at least 25% of the plurality of modified Tcells expresses one or more cell-surface marker(s) of a stem memory Tcell (T_(SCM)), thereby producing a plurality of modified stem memory Tcells (T_(SCM)). In certain embodiments, the method produces a pluralityof modified T cells, wherein at least 50% of the plurality of modified Tcells expresses one or more cell-surface marker(s) of a stem memory Tcell (T_(SCM)), thereby producing a plurality of modified stem memory Tcells (T_(SCM)). In certain embodiments, the method produces a pluralityof modified T cells, wherein at least 60% of the plurality of modified Tcells expresses one or more cell-surface marker(s) of a stem memory Tcell (T_(SCM)), thereby producing a plurality of modified stem memory Tcells (T_(SCM)). In certain embodiments, the method produces a pluralityof modified T cells, wherein at least 75% of the plurality of modified Tcells expresses one or more cell-surface marker(s) of a stem memory Tcell (T_(SCM)), thereby producing a plurality of modified stem memory Tcells (T_(SCM)). In certain embodiments, the method produces a pluralityof modified T cells, wherein at least 80% of the plurality of modified Tcells expresses one or more cell-surface marker(s) of a stem memory Tcell (T_(SCM)), thereby producing a plurality of modified stem memory Tcells (T_(SCM)). In certain embodiments, the method produces a pluralityof modified T cells, wherein at least 85% of the plurality of modified Tcells expresses one or more cell-surface marker(s) of a stem memory Tcell (T_(SCM)), thereby producing a plurality of modified stem memory Tcells (T_(SCM)). In certain embodiments, the method produces a pluralityof modified T cells, wherein at least 90% of the plurality of modified Tcells expresses one or more cell-surface marker(s) of a stem memory Tcell (T_(SCM)), thereby producing a plurality of modified stem memory Tcells (T_(SCM)). In certain embodiments, the method produces a pluralityof modified T cells, wherein at least 95% of the plurality of modified Tcells expresses one or more cell-surface marker(s) of a stem memory Tcell (T_(SCM)), thereby producing a plurality of modified stem memory Tcells (T_(SCM)). In certain embodiments, the cell-surface markerscomprise CD62L and CD45RA. In certain embodiments, the cell-surfacemarkers of the CAR-T_(SCM) comprise one or more of CD62L, CD45RA, CD28,CCR7, CD127, CD45RO, CD95, CD95 and IL-2Rβ. In certain embodiments, thecell-surface markers of the CAR-T_(SCM) comprise one or more of CD45RA,CD95, IL-2Rβ, CR7, and CD62L. In certain embodiments of this method, thetransposon is a Helraiser transposon. In certain embodiments, and, inparticular, those embodiments wherein the transposon is a Helraisertransposon, the transposase is a Helitron transposase.

In certain embodiments of the methods of the disclosure, the transposaseis a Helitron transposase. Helitron transposases mobilize the Helraisertransposon, an ancient element from the bat genome that was active about30 to 36 million years ago. An exemplary Helraiser transposon of thedisclosure includes Helibat1, which comprises a nucleic acid sequencecomprising:

(SEQ ID NO: 27)   1 TCCTATATAA TAAAAGAGAA ACATGCAAAT TGACCATCCC TCCGCTACGC TCAAGCCACG  61 CCCACCAGCC AATCAGAAGT GACTATGCAA ATTAACCCAA CAAAGATGGC AGTTAAATTT 121 GCATACGCAG GTGTCAAGCG CCCCAGGAGG CAACGGCGGC CGCGGGCTCC CAGGACCTTC 181 GCTGGCCCCG GGAGGCGAGG CCGGCCGCGC CTAGCCACAC CCGCGGGCTC CCGGGACCTT 241 CGCCAGCAGA GAGCAGAGCG GGAGAGCGGG CGGAGAGCGG GAGGTTTGGA GGACTTGGCA 301 GAGCAGGAGG CCGCTGGACA TAGAGCAGAG CGAGAGAGAG GGTGGCTTGG AGGGCGTGGC 361 TCCCTCTGTC ACCCCAGCTT CCTCATCACA GCTGTGGAAA CTGACAGCAG GGAGGAGGAA 421 GTCCCACCCC CACAGAATCA GCCAGAATCA GCCGTTGGTC AGACAGCTCT CAGCGGCCTG 481 ACAGCCAGGA CTCTCATTCA CCTGCATCTC AGACCGTGAC AGTAGAGAGG TGGGACTATG 541 TCTAAAGAAC AACTGTTGAT ACAACGTAGC TCTGCAGCCG AAAGATGCCG GCGTTATCGA 601 CAGAAAATGT CTGCAGAGCA ACGTGCGTCT GATCTTGAAA GAAGGCGGCG CCTGCAACAG 661 AATGTATCTG AAGAGCAGCT ACTGGAAAAA CGTCGCTCTG AAGCCGAAAA ACAGCGGCGT 721 CATCGACAGA AAATGTCTAA AGACCAACGT GCCTTTGAAG TTGAAAGAAG GCGGTGGCGA 781 CGACAGAATA TGTCTAGAGA ACAGTCATCA ACAAGTACTA CCAATACCGG TAGGAACTGC 841 CTTCTCAGCA AAAATGGAGT ACATGAGGAT GCAATTCTCG AACATAGTTG TGGTGGAATG 901 ACTGTTCGAT GTGAATTTTG CCTATCACTA AATTTCTCTG ATGAAAAACC ATCCGATGGG 961 AAATTTACTC GATGTTGTAG CAAAGGGAAA GTCTGTCCAA ATGATATACA TTTTCCAGAT1021 TACCCGGCAT ATTTAAAAAG ATTAATGACA AACGAAGATT CTGACAGTAA AAATTTCATG1081 GAAAATATTC GTTCCATAAA TAGTTCTTTT GCTTTTGCTT CCATGGGTGC AAATATTGCA1141 TCGCCATCAG GATATGGGCC ATACTGTTTT AGAATACACG GACAAGTTTA TCACCGTACT1201 GGAACTTTAC ATCCTTCGGA TGGTGTTTCT CGGAAGTTTG CTCAACTCTA TATTTTGGAT1261 ACAGCCGAAG CTACAAGTAA AAGATTAGCA ATGCCAGAAA ACCAGGGCTG CTCAGAAAGA1321 CTCATGATCA ACATCAACAA CCTCATGCAT GAAATAAATG AATTAACAAA ATCGTACAAG1381 ATGCTACATG AGGTAGAAAA GGAAGCCCAA TCTGAAGCAG CAGCAAAAGG TATTGCTCCC1441 ACAGAAGTAA CAATGGCGAT TAAATACGAT CGTAACAGTG ACCCAGGTAG ATATAATTCT1501 CCCCGTGTAA CCGAGGTTGC TGTCATATTC AGAAACGAAG ATGGAGAACC TCCTTTTGAA1561 AGGGACTTGC TCATTCATTG TAAACCAGAT CCCAATAATC CAAATGCCAC TAAAATGAAA1621 CAAATCAGTA TCCTGTTTCC TACATTAGAT GCAATGACAT ATCCTATTCT TTTTCCACAT1681 GGTGAAAAAG GCTGGGGAAC AGATATTGCA TTAAGACTCA GAGACAACAG TGTAATCGAC1741 AATAATACTA GACAAAATGT AAGGACACGA GTCACACAAA TGCAGTATTA TGGATTTCAT1801 CTCTCTGTGC GGGACACGTT CAATCCTATT TTAAATGCAG GAAAATTAAC TCAACAGTTT1861 ATTGTGGATT CATATTCAAA AATGGAGGCC AATCGGATAA ATTTCATCAA AGCAAACCAA1921 TCTAAGTTGA GAGTTGAAAA ATATAGTGGT TTGATGGATT ATCTCAAATC TAGATCTGAA1981 AATGACAATG TGCCGATTGG TAAAATGATA ATACTTCCAT CATCTTTTGA GGGTAGTCCC2041 AGAAATATGC AGCAGCGATA TCAGGATGCT ATGGCAATTG TAACGAAGTA TGGCAAGCCC2101 GATTTATTCA TAACCATGAC ATGCAACCCC AAATGGGCAG ATATTACAAA CAATTTACAA2161 CGCTGGCAAA AAGTTGAAAA CAGACCTGAC TTGGTAGCCA GAGTTTTTAA TATTAAGCTG2221 AATGCTCTTT TAAATGATAT ATGTAAATTC CATTTATTTG GCAAAGTAAT AGCTAAAATT2281 CATGTCATTG AATTTCAGAA ACGCGGACTG CCTCACGCTC ACATATTATT GATATTAGAT2341 AGTGAGTCCA AATTACGTTC AGAAGATGAC ATTGACCGTA TAGTTAAGGC AGAAATTCCA2401 GATGAAGACC AGTGTCCTCG ACTTTTTCAA ATTGTAAAAT CAAATATGGT ACATGGACCA2461 TGTGGAATAC AAAATCCAAA TAGTCCATGT ATGGAAAATG GAAAATGTTC AAAGGGATAT2521 CCAAAAGAAT TTCAAAATGC GACCATTGGA AATATTGATG GATATCCCAA ATACAAACGA2581 AGATCTGGTA GCACCATGTC TATTGGAAAT AAAGTTGTCG ATAACACTTG GATTGTCCCT2641 TATAACCCGT ATTTGTGCCT TAAATATAAC TGTCATATAA ATGTTGAAGT CTGTGCATCA2701 ATTAAAAGTG TCAAATATTT ATTTAAATAC ATCTATAAAG GGCACGATTG TGCAAATATT2761 CAAATTTCTG AAAAAAATAT TATCAATCAT GACGAAGTAC AGGACTTCAT TGACTCCAGG2821 TATGTGAGCG CTCCTGAGGC TGTTTGGAGA CTTTTTGCAA TGCGAATGCA TGACCAATCT2881 CATGCAATCA CAAGATTAGC TATTCATTTG CCAAATGATC AGAATTTGTA TTTTCATACC2941 GATGATTTTG CTGAAGTTTT AGATAGGGCT AAAAGGCATA ACTCGACTTT GATGGCTTGG3001 TTCTTATTGA ATAGAGAAGA TTCTGATGCA CGTAATTATT ATTATTGGGA GATTCCACAG3061 CATTATGTGT TTAATAATTC TTTGTGGACA AAACGCCGAA AGGGTGGGAA TAAAGTATTA3121 GGTAGACTGT TCACTGTGAG CTTTAGAGAA CCAGAACGAT ATTACCTTAG ACTTTTGCTT3181 CTGCATGTAA AAGGTGCGAT AAGTTTTGAG GATCTGCGAA CTGTAGGAGG TGTAACTTAT3241 GATACATTTC ATGAAGCTGC TAAACACCGA GGATTATTAC TTGATGACAC TATCTGGAAA3301 GATACGATTG ACGATGCAAT CATCCTTAAT ATGCCCAAAC AACTACGGCA ACTTTTTGCA3361 TATATATGTG TGTTTGGATG TCCTTCTGCT GCAGACAAAT TATGGGATGA GAATAAATCT3421 CATTTTATTG AAGATTTCTG TTGGAAATTA CACCGAAGAG AAGGTGCCTG TGTGAACTGT3481 GAAATGCATG CCCTTAACGA AATTCAGGAG GTATTCACAT TGCATGGAAT GAAATGTTCA3541 CATTTCAAAC TTCCGGACTA TCCTTTATTA ATGAATGCAA ATACATGTGA TCAATTGTAC3601 GAGCAACAAC AGGCAGAGGT TTTGATAAAT TCTCTGAATG ATGAACAGTT GGCAGCCTTT3661 CAGACTATAA CTTCAGCCAT CGAAGATCAA ACTGTACACC CCAAATGCTT TTTCTTGGAT3721 GGTCCAGGTG GTAGTGGAAA AACATATCTG TATAAAGTTT TAACACATTA TATTAGAGGT3781 CGTGGTGGTA CTGTTTTACC CACAGCATCT ACAGGAATTG CTGCAAATTT ACTTCTTGGT3841 GGAAGAACCT TTCATTCCCA ATATAAATTA CCAATTCCAT TAAATGAAAC TTCAATTTCT3901 AGACTCGATA TAAAGAGTGA AGTTGCTAAA ACCATTAAAA AGGCCCAACT TCTCATTATT3961 GATGAATGCA CCATGGCATC CAGTCATGCT ATAAACGCCA TAGATAGATT ACTAAGAGAA4021 ATTATGAATT TGAATGTTGC ATTTGGTGGG AAAGTTCTCC TTCTCGGAGG GGATTTTCGA4081 CAATGTCTCA GTATTGTACC ACATGCTATG CGATCGGCCA TAGTACAAAC GAGTTTAAAG4141 TACTGTAATG TTTGGGGATG TTTCAGAAAG TTGTCTCTTA AAACAAATAT GAGATCAGAG4201 GATTCTGCTT ATAGTGAATG GTTAGTAAAA CTTGGAGATG GCAAACTTGA TAGCAGTTTT4261 CATTTAGGAA TGGATATTAT TGAAATCCCC CATGAAATGA TTTGTAACGG ATCTATTATT4321 GAAGCTACCT TTGGAAATAG TATATCTATA GATAATATTA AAAATATATC TAAACGTGCA4381 ATTCTTTGTC CAAAAAATGA GCATGTTCAA AAATTAAATG AAGAAATTTT GGATATACTT4441 GATGGAGATT TTCACACATA TTTGAGTGAT GATTCCATTG ATTCAACAGA TGATGCTGAA4501 AAGGAAAATT TTCCCATCGA ATTTCTTAAT AGTATTACTC CTTCGGGAAT GCCGTGTCAT4561 AAATTAAAAT TGAAAGTGGG TGCAATCATC ATGCTATTGA GAAATCTTAA TAGTAAATGG4621 GGTCTTTGTA ATGGTACTAG ATTTATTATC AAAAGATTAC GACCTAACAT TATCGAAGCT4681 GAAGTATTAA CAGGATCTGC AGAGGGAGAG GTTGTTCTGA TTCCAAGAAT TGATTTGTCC4741 CCATCTGACA CTGGCCTCCC ATTTAAATTA ATTCGAAGAC AGTTTCCCGT GATGCCAGCA4801 TTTGCGATGA CTATTAATAA ATCACAAGGA CAAACTCTAG ACAGAGTAGG AATATTCCTA4861 CCTGAACCCG TTTTCGCACA TGGTCAGTTA TATGTTGCTT TCTCTCGAGT TCGAAGAGCA4921 TGTGACGTTA AAGTTAAAGT TGTAAATACT TCATCACAAG GGAAATTAGT CAAGCACTCT4981 GAAAGTGTTT TTACTCTTAA TGTGGTATAC AGGGAGATAT TAGAATAAGT TTAATCACTT5041 TATCAGTCAT TGTTTGCATC AATGTTGTTT TTATATCATG TTTTTGTTGT TTTTATATCA5101 TGTCTTTGTT GTTGTTATAT CATGTTGTTA TTGTTTATTT ATTAATAAAT TTATGTATTA5161 TTTTCATATA CATTTTACTC ATTTCCTTTC ATCTCTCACA CTTCTATTAT AGAGAAAGGG5221 CAAATAGCAA TATTAAAATA TTTCCTCTAA TTAATTCCCT TTCAATGTGC ACGAATTTCG5281 TGCACCGGGC CACTAG.

Unlike other transposases, the Helitron transposase does not contain anRNase-H like catalytic domain, but instead comprises a RepHel motif madeup of a replication initiator domain (Rep) and a DNA helicase domain.The Rep domain is a nuclease domain of the HUH superfamily of nucleases.

An exemplary Helitron transposase of the disclosure comprises an aminoacid sequence comprising:

(SEQ ID NO: 28)   1 MSKEQLLIQR SSAAERCRRY RQKMSAEQRA SDLERRRRLQ QNVSEEQLLE KRRSEAEKQR  61 RHRQKMSKDQ RAFEVERRRW RRQNMSREQS STSTTNTGRN CLLSKNGVHE DAILEHSCGG 121 MTVRCEFCLS LNFSDEKPSD GKFTRCCSKG KVCPNDIHFP DYPAYLKRLM TNEDSDSKNF 181 MENIRSINSS FAFASMGANI ASPSGYGPYC FRIHGQVYHR TGTLHPSDGV SRKFAQLYIL 241 DTAEATSKRL AMPENQGCSE RLMININNLM HEINELTKSY KMLHEVEKEA QSEAAAKGIA 301 PTEVTMAIKY DRNSDPGRYN SPRVTEVAVI FRNEDGEPPF ERDLLIHCKP DPNNPNATKM 361 KQISILFPTL DAMTYPILFP HGEKGWGTDI ALRLRDNSVI DNNTRQNVRT RVTQMQYYGF 421 HLSVRDTFNP ILNAGKLTQQ FIVDSYSKME ANRINFIKAN QSKLRVEKYS GLMDYLKSRS 481 ENDNVPIGKM IILPSSFEGS PRNMQQRYQD AMAIVTKYGK PDLFITMTCN PKWADITNNL 541 QRWQKVENRP DLVARVFNIK LNALLNDICK FHLFGKVIAK IHVIEFQKRG LPHAHILLIL 601 DSESKLRSED DIDRIVKAEI PDEDQCPRLF QIVKSNMVHG PCGIQNPNSP CMENGKCSKG 661 YPKEFQNATI GNIDGYPKYK RRSGSTMSIG NKVVDNTWIV PYNPYLCLKY NCHINVEVCA 721 SIKSVKYLFK YIYKGHDCAN IQISEKNIIN HDEVQDFIDS RYVSAPEAVW RLFAMRMHDQ 781 SHAITRLAIH LPNDQNLYFH TDDFAEVLDR AKRHNSTLMA WFLLNREDSD ARNYYYWEIP 841 QHYVFNNSLW TKRRKGGNKV LGRLFTVSFR EPERYYLRLL LLHVKGAISF EDLRTVGGVT 901 YDTFHEAAKH RGLLLDDTIW KDTIDDAIIL NMPKQLRQLF AYICVFGCPS AADKLWDENK 961 SHFIEDFCWK LHRREGACVN CEMHALNEIQ EVFTLHGMKC SHFKLPDYPL LMNANTCDQL1021 YEQQQAEVLI NSLNDEQLAA FQTITSAIED QTVHPKCFFL DGPGGSGKTY LYKVLTHYIR1081 GRGGTVLPTA STGIAANLLL GGRTFHSQYK LPIPLNETSI SRLDIKSEVA KTIKKAQLLI1141 IDECTMASSH AINAIDRLLR EIMNLNVAFG GKVLLLGGDF RQCLSIVPHA MRSAIVQTSL1201 KYCNVWGCFR KLSLKTNMRS EDSAYSEWLV KLGDGKLDSS FHLGMDIIEI PHEMICNGSI1261 IEATFGNSIS IDNIKNISKR AILCPKNEHV QKLNEEILDI LDGDFHTYLS DDSIDSTDDA1321 EKENFPIEFL NSITPSGMPC HKLKLKVGAI IMLLRNLNSK WGLCNGTRFI IKRLRPNIIE1381 AEVLTGSAEG EVVLIPRIDL SPSDTGLPFK LIRRQFPVMP AFAMTINKSQ GQTLDRVGIF1441 LPEPVFAHGQ LYVAFSRVRR ACDVKVKVVN TSSQGKLVKH SESVFTLNVV YREILE.

In Helitron transpositions, a hairpin close to the 3′ end of thetransposon functions as a terminator. However, this hairpin can bebypassed by the transposase, resulting in the transduction of flankingsequences. In addition, Helraiser transposition generates covalentlyclosed circular intermediates. Furthermore, Helitron transpositions canlack target site duplications. In the Helraiser sequence, thetransposase is flanked by left and right terminal sequences termed LTSand RTS. These sequences terminate with a conserved 5′-TC/CTAG-3′ motif.A 19 bp palindromic sequence with the potential to form the hairpintermination structure is located 11 nucleotides upstream of the RTS andconsists of the sequence GTGCACGAATTTCGTGCACCGGGCCACTAG (SEQ ID NO: 29).

The disclosure provides a method of producing a modified stem memory Tcell (T_(SCM)), comprising introducing into a primary human T cell (a) atransposon composition comprising a transposon comprising an antigenreceptor or a therapeutic protein and (b) a transposase compositioncomprising a transposase or a sequence encoding the transposase; toproduce a modified T cell, wherein the modified T cell expresses one ormore cell-surface marker(s) of a stem memory T cell (T_(SCM)), therebyproducing a modified stem memory T cell (T_(SCM)). The disclosureprovides a method of producing a plurality of modified stem memory Tcells (T_(SCM)), comprising introducing into a plurality of primaryhuman T cell (a) a transposon composition comprising a transposoncomprising an antigen receptor and (b) a transposase compositioncomprising a transposase or a sequence encoding the transposase; toproduce a plurality of modified T cells, wherein at least 2%, 5%, 10%,15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 95%, 99% or any percentage in between of the plurality of modifiedT cells expresses one or more cell-surface marker(s) of a stem memory Tcell (T_(SCM)), thereby producing a plurality of modified stem memory Tcells (T_(SCM)). In certain embodiments, the method produces a pluralityof modified T cells, wherein at least 25% of the plurality of modified Tcells expresses one or more cell-surface marker(s) of a stem memory Tcell (T_(SCM)), thereby producing a plurality of modified stem memory Tcells (T_(SCM)). In certain embodiments, the method produces a pluralityof modified T cells, wherein at least 50% of the plurality of modified Tcells expresses one or more cell-surface marker(s) of a stem memory Tcell (T_(SCM)), thereby producing a plurality of modified stem memory Tcells (T_(SCM)). In certain embodiments, the method produces a pluralityof modified T cells, wherein at least 60% of the plurality of modified Tcells expresses one or more cell-surface marker(s) of a stem memory Tcell (T_(SCM)), thereby producing a plurality of modified stem memory Tcells (T_(SCM)). In certain embodiments, the method produces a pluralityof modified T cells, wherein at least 75% of the plurality of modified Tcells expresses one or more cell-surface marker(s) of a stem memory Tcell (T_(SCM)), thereby producing a plurality of modified stem memory Tcells (T_(SCM)). In certain embodiments, the method produces a pluralityof modified T cells, wherein at least 80% of the plurality of modified Tcells expresses one or more cell-surface marker(s) of a stem memory Tcell (T_(SCM)), thereby producing a plurality of modified stem memory Tcells (T_(SCM)). In certain embodiments, the method produces a pluralityof modified T cells, wherein at least 85% of the plurality of modified Tcells expresses one or more cell-surface marker(s) of a stem memory Tcell (T_(SCM)), thereby producing a plurality of modified stem memory Tcells (T_(SCM)). In certain embodiments, the method produces a pluralityof modified T cells, wherein at least 90% of the plurality of modified Tcells expresses one or more cell-surface marker(s) of a stem memory Tcell (T_(SCM)), thereby producing a plurality of modified stem memory Tcells (T_(SCM)). In certain embodiments, the method produces a pluralityof modified T cells, wherein at least 95% of the plurality of modified Tcells expresses one or more cell-surface marker(s) of a stem memory Tcell (T_(SCM)), thereby producing a plurality of modified stem memory Tcells (T_(SCM)). In certain embodiments, the cell-surface markerscomprise CD62L and CD45RA. In certain embodiments, the cell-surfacemarkers of the CAR-T_(SCM) comprise one or more of CD62L, CD45RA, CD28,CCR7, CD127, CD45RO, CD95, CD95 and IL-2Rβ. In certain embodiments, thecell-surface markers of the CAR-T_(SCM) comprise one or more of CD45RA,CD95, IL-2Rβ, CR7, and CD62L. In certain embodiments of this method, thetransposon is a Tol2 transposon. In certain embodiments, including thoseembodiments wherein the transposon is a Tol2 transposon, the transposaseis a Tol2 transposase.

In certain embodiments of the methods of the disclosure, the transposaseis a Tol2 transposase. Tol2 transposons may be isolated or derived fromthe genome of the medaka fish, and may be similar to transposons of thehAT family. Exemplary Tol2 transposons of the disclosure are encoded bya sequence comprising about 4.7 kilobases and contain a gene encodingthe Tol2 transposase, which contains four exons. An exemplary Tol2transposase of the disclosure comprises an amino acid sequencecomprising the following:

(SEQ ID NO: 30)  1 MEEVCDSSAA ASSTVQNQPQ DQEHPWPYLR EFFSLSGVNK DSFKMKCVLC LPLNKEISAF 61 KSSPSNLRKH IERMHPNYLK NYSKLTAQKR KIGTSTHASS SKQLKVDSVF PVKHVSPVTV121 NKAILRYIIQ GLHPFSTVDL PSFKELISTL QPGISVITRP TLRSKIAEAA LIMKQKVTAA181 MSEVEWIATT TDCWTARRKS FIGVTAHWIN PGSLERHSAA LACKRLMGSH TFEVLASAMN241 DIHSEYEIRD KVVCTTTDSG SNFMKAFRVF GVENNDIETE ARRCESDDTD SEGCGEGSDG301 VEFQDASRVL DQDDGFEFQL PKHQKCACHL LNLVSSVDAQ KALSNEHYKK LYRSVFGKCQ361 ALWNKSSRSA LAAEAVESES RLQLLRPNQT RWNSTFMAVD RILQICKEAG EGALRNICTS421 LEVPMFNPAE MLFLTEWANT MRPVAKVLDI LQAETNTQLG WLLPSVHQLS LKLQRLHHSL481 RYCDPLVDAL QQGIQTRFKH MFEDPEIIAA AILLPKFRTS WTNDETIIKR GMDYIRVHLE541 PLDHKKELAN SSSDDEDFFA SLKPTTHEAS KELDGYLACV SDTRESLLTF PAICSLSIKT601 NTPLPASAAC ERLFSTAGLL FSPKRARLDT NNFENQLLLK LNLRFYNFE.

An exemplary Tol2 transposon of the disclosure, including invertedrepeats, subterminal sequences and the Tol2 transposase, is encoded by anucleic acid sequence comprising the following:

(SEQ ID NO: 31)   1 CAGAGGTGTA AAGTACTTGA GTAATTTTAC TTGATTACTG TACTTAAGTA TTATTTTTGG  61 GGATTTTTAC TTTACTTGAG TACAATTAAA AATCAATACT TTTACTTTTA CTTAATTACA 121 TTTTTTTAGA AAAAAAAGTA CTTTTTACTC CTTACAATTT TATTTACAGT CAAAAAGTAC 181 TTATTTTTTG GAGATCACTT CATTCTATTT TCCCTTGCTA TTACCAAACC AATTGAATTG 241 CGCTGATGCC CAGTTTAATT TAAATGTTAT TTATTCTGCC TATGAAAATC GTTTTCACAT 301 TATATGAAAT TGGTCAGACA TGTTCATTGG TCCTTTGGAA GTGACGTCAT GTCACATCTA 361 TTACCACAAT GCACAGCACC TTGACCTGGA AATTAGGGAA ATTATAACAG TCAATCAGTG 421 GAAGAAAATG GAGGAAGTAT GTGATTCATC AGCAGCTGCG AGCAGCACAG TCCAAAATCA 481 GCCACAGGAT CAAGAGCACC CGTGGCCGTA TCTTCGCGAA TTCTTTTCTT TAAGTGGTGT 541 AAATAAAGAT TCATTCAAGA TGAAATGTGT CCTCTGTCTC CCGCTTAATA AAGAAATATC 601 GGCCTTCAAA AGTTCGCCAT CAAACCTAAG GAAGCATATT GAGGTAAGTA CATTAAGTAT 661 TTTGTTTTAC TGATAGTTTT TTTTTTTTTT TTTTTTTTTT TTTTTGGGTG TGCATGTTTT 721 GACGTTGATG GCGCGCCTTT TATATGTGTA GTAGGCCTAT TTTCACTAAT GCATGCGATT 781 GACAATATAA GGCTCACGTA ATAAAATGCT AAAATGCATT TGTAATTGGT AACGTTAGGT 841 CCACGGGAAA TTTGGCGCCT ATTGCAGCTT TGAATAATCA TTATCATTCC GTGCTCTCAT 901 TGTGTTTGAA TTCATGCAAA ACACAAGAAA ACCAAGCGAG AAATTTTTTT CCAAACATGT 961 TGTATTGTCA AAACGGTAAC ACTTTACAAT GAGGTTGATT AGTTCATGTA TTAACTAACA1021 TTAAATAACC ATGAGCAATA CATTTGTTAC TGTATCTGTT AATCTTTGTT AACGTTAGTT1081 AATAGAAATA CAGATGTTCA TTGTTTGTTC ATGTTAGTTC ACAGTGCATT AACTAATGTT1141 AACAAGATAT AAAGTATTAG TAAATGTTGA AATTAACATG TATACGTGCA GTTCATTATT1201 AGTTCATGTT AACTAATGTA GTTAACTAAC GAACCTTATT GTAAAAGTGT TACCATCAAA1261 ACTAATGTAA TGAAATCAAT TCACCCTGTC ATGTCAGCCT TACAGTCCTG TGTTTTTGTC1321 AATATAATCA GAAATAAAAT TAATGTTTGA TTGTCACTAA ATGCTACTGT ATTTCTAAAA1381 TCAACAAGTA TTTAACATTA TAAAGTGTGC AATTGGCTGC AAATGTCAGT TTTATTAAAG1441 GGTTAGTTCA CCCAAAAATG AAAATAATGT CATTAATGAC TCGCCCTCAT GTCGTTCCAA1501 GCCCGTAAGA CCTCCGTTCA TCTTCAGAAC ACAGTTTAAG ATATTTTAGA TTTAGTCCGA1561 GAGCTTTCTG TGCCTCCATT GAGAATGTAT GTACGGTATA CTGTCCATGT CCAGAAAGGT1621 AATAAAAACA TCAAAGTAGT CCATGTGACA TCAGTGGGTT AGTTAGAATT TTTTGAAGCA1681 TCGAATACAT TTTGGTCCAA AAATAACAAA ACCTACGACT TTATTCGGCA TTGTATTCTC1741 TTCCGGGTCT GTTGTCAATC CGCGTTCACG ACTTCGCAGT GACGCTACAA TGCTGAATAA1801 AGTCGTAGGT TTTGTTATTT TTGGACCAAA ATGTATTTTC GATGCTTCAA ATAATTCTAC1861 CTAACCCACT GATGTCACAT GGACTACTTT GATGTTTTTA TTACCTTTCT GGACATGGAC1921 AGTATACCGT ACATACATTT TCAGTGGAGG GACAGAAAGC TCTCGGACTA AATCTAAAAT1981 ATCTTAAACT GTGTTCCGAA GATGAACGGA GGTGTTACGG GCTTGGAACG ACATGAGGGT2041 GAGTCATTAA TGACATCTTT TCATTTTTGG GTGAACTAAC CCTTTAATGC TGTAATCAGA2101 GAGTGTATGT GTAATTGTTA CATTTATTGC ATACAATATA AATATTTATT TGTTGTTTTT2161 ACAGAGAATG CACCCAAATT ACCTCAAAAA CTACTCTAAA TTGACAGCAC AGAAGAGAAA2221 GATCGGGACC TCCACCCATG CTTCCAGCAG TAAGCAACTG AAAGTTGACT CAGTTTTCCC2281 AGTCAAACAT GTGTCTCCAG TCACTGTGAA CAAAGCTATA TTAAGGTACA TCATTCAAGG2341 ACTTCATCCT TTCAGCACTG TTGATCTGCC ATCATTTAAA GAGCTGATTA GTACACTGCA2401 GCCTGGCATT TCTGTCATTA CAAGGCCTAC TTTACGCTCC AAGATAGCTG AAGCTGCTCT2461 GATCATGAAA CAGAAAGTGA CTGCTGCCAT GAGTGAAGTT GAATGGATTG CAACCACAAC2521 GGATTGTTGG ACTGCACGTA GAAAGTCATT CATTGGTGTA ACTGCTCACT GGATCAACCC2581 TGGAAGTCTT GAAAGACATT CCGCTGCACT TGCCTGCAAA AGATTAATGG GCTCTCATAC2641 TTTTGAGGTA CTGGCCAGTG CCATGAATGA TATCCACTCA GAGTATGAAA TACGTGACAA2701 GGTTGTTTGC ACAACCACAG ACAGTGGTTC CAACTTTATG AAGGCTTTCA GAGTTTTTGG2761 TGTGGAAAAC AATGATATCG AGACTGAGGC AAGAAGGTGT GAAAGTGATG ACACTGATTC2821 TGAAGGCTGT GGTGAGGGAA GTGATGGTGT GGAATTCCAA GATGCCTCAC GAGTCCTGGA2881 CCAAGACGAT GGCTTCGAAT TCCAGCTACC AAAACATCAA AAGTGTGCCT GTCACTTACT2941 TAACCTAGTC TCAAGCGTTG ATGCCCAAAA AGCTCTCTCA AATGAACACT ACAAGAAACT3001 CTACAGATCT GTCTTTGGCA AATGCCAAGC TTTATGGAAT AAAAGCAGCC GATCGGCTCT3061 AGCAGCTGAA GCTGTTGAAT CAGAAAGCCG GCTTCAGCTT TTAAGGCCAA ACCAAACGCG3121 GTGGAATTCA ACTTTTATGG CTGTTGACAG AATTCTTCAA ATTTGCAAAG AAGCAGGAGA3181 AGGCGCACTT CGGAATATAT GCACCTCTCT TGAGGTTCCA ATGTAAGTGT TTTTCCCCTC3241 TATCGATGTA AACAAATGTG GGTTGTTTTT GTTTAATACT CTTTGATTAT GCTGATTTCT3301 CCTGTAGGTT TAATCCAGCA GAAATGCTGT TCTTGACAGA GTGGGCCAAC ACAATGCGTC3361 CAGTTGCAAA AGTACTCGAC ATCTTGCAAG CGGAAACGAA TACACAGCTG GGGTGGCTGC3421 TGCCTAGTGT CCATCAGTTA AGCTTGAAAC TTCAGCGACT CCACCATTCT CTCAGGTACT3481 GTGACCCACT TGTGGATGCC CTACAACAAG GAATCCAAAC ACGATTCAAG CATATGTTTG3541 AAGATCCTGA GATCATAGCA GCTGCCATCC TTCTCCCTAA ATTTCGGACC TCTTGGACAA3601 ATGATGAAAC CATCATAAAA CGAGGTAAAT GAATGCAAGC AACATACACT TGACGAATTC3661 TAATCTGGGC AACCTTTGAG CCATACCAAA ATTATTCTTT TATTTATTTA TTTTTGCACT3721 TTTTAGGAAT GTTATATCCC ATCTTTGGCT GTGATCTCAA TATGAATATT GATGTAAAGT3781 ATTCTTGCAG CAGGTTGTAG TTATCCCTCA GTGTTTCTTG AAACCAAACT CATATGTATC3841 ATATGTGGTT TGGAAATGCA GTTAGATTTT ATGCTAAAAT AAGGGATTTG CATGATTTTA3901 GATGTAGATG ACTGCACGTA AATGTAGTTA ATGACAAAAT CCATAAAATT TGTTCCCAGT3961 CAGAAGCCCC TCAACCAAAC TTTTCTTTGT GTCTGCTCAC TGTGCTTGTA GGCATGGACT4021 ACATCAGAGT GCATCTGGAG CCTTTGGACC ACAAGAAGGA ATTGGCCAAC AGTTCATCTG4081 ATGATGAAGA TTTTTTCGCT TCTTTGAAAC CGACAACACA TGAAGCCAGC AAAGAGTTGG4141 ATGGATATCT GGCCTGTGTT TCAGACACCA GGGAGTCTCT GCTCACGTTT CCTGCTATTT4201 GCAGCCTCTC TATCAAGACT AATACACCTC TTCCCGCATC GGCTGCCTGT GAGAGGCTTT4261 TCAGCACTGC AGGATTGCTT TTCAGCCCCA AAAGAGCTAG GCTTGACACT AACAATTTTG4321 AGAATCAGCT TCTACTGAAG TTAAATCTGA GGTTTTACAA CTTTGAGTAG CGTGTACTGG4381 CATTAGATTG TCTGTCTTAT AGTTTGATAA TTAAATACAA ACAGTTCTAA AGCAGGATAA4441 AACCTTGTAT GCATTTCATT TAATGTTTTT TGAGATTAAA AGCTTAAACA AGAATCTCTA4501 GTTTTCTTTC TTGCTTTTAC TTTTACTTCC TTAATACTCA AGTACAATTT TAATGGAGTA4561 CTTTTTTACT TTTACTCAAG TAAGATTCTA GCCAGATACT TTTACTTTTA ATTGAGTAAA4621 ATTTTCCCTA AGTACTTGTA CTTTCACTTG AGTAAAATTT TTGAGTACTT TTTACACCTC4681 TG.

The disclosure provides a method of producing a modified central memoryT-cell (T_(CM)), comprising introducing into a primary human T cell (a)a transposon composition comprising a transposon comprising an antigenreceptor or a therapeutic protein and (b) a transposase compositioncomprising a transposase or a sequence encoding the transposase; toproduce a modified T cell, wherein the modified T cell expresses one ormore cell-surface marker(s) of a central memory T-cell (T_(CM)), therebyproducing a modified central memory T-cell (T_(CM)). The disclosureprovides a method of producing a plurality of modified central memoryT-cells (T_(CM)), comprising introducing into a plurality of primaryhuman T cell (a) a transposon composition comprising a transposoncomprising an antigen receptor and (b) a transposase compositioncomprising a transposase or a sequence encoding the transposase; toproduce a plurality of modified T cells, wherein at least 2%, 5%, 10%,15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 95%, 99% or any percentage in between of the plurality of modifiedT cells expresses one or more cell-surface marker(s) of a central memoryT-cell (T_(CM)), thereby producing a plurality of modified centralmemory T-cells (T_(CM)). In certain embodiments, the method produces aplurality of modified T cells, wherein at least 25% of the plurality ofmodified T cells expresses one or more cell-surface marker(s) of centralmemory T-cell (T_(CM)), thereby producing a plurality of modifiedcentral memory T-cells (T_(CM)). In certain embodiments, the methodproduces a plurality of modified T cells, wherein at least 50% of theplurality of modified T cells expresses one or more cell-surfacemarker(s) of central memory T-cell (T_(CM)), thereby producing aplurality of modified central memory T-cells (T_(CM)). In certainembodiments, the method produces a plurality of modified T cells,wherein at least 60% of the plurality of modified T cells expresses oneor more cell-surface marker(s) of central memory T-cell (T_(CM)),thereby producing a plurality of modified central memory T-cells(T_(CM)). In certain embodiments, the method produces a plurality ofmodified T cells, wherein at least 75% of the plurality of modified Tcells expresses one or more cell-surface marker(s) of central memoryT-cell (T_(CM)), thereby producing a plurality of modified centralmemory T-cells (T_(CM)). In certain embodiments, the method produces aplurality of modified T cells, wherein at least 80% of the plurality ofmodified T cells expresses one or more cell-surface marker(s) of centralmemory T-cell (T_(CM)), thereby producing a plurality of modifiedcentral memory T-cells (T_(CM)). In certain embodiments, the methodproduces a plurality of modified T cells, wherein at least 85% of theplurality of modified T cells expresses one or more cell-surfacemarker(s) of central memory T-cell (T_(CM)), thereby producing aplurality of modified central memory T-cells (T_(CM)). In certainembodiments, the method produces a plurality of modified T cells,wherein at least 90% of the plurality of modified T cells expresses oneor more cell-surface marker(s) of central memory T-cell (T_(CM)),thereby producing a plurality of modified central memory T-cells(T_(CM)). In certain embodiments, the method produces a plurality ofmodified T cells, wherein at least 95% of the plurality of modified Tcells expresses one or more cell-surface marker(s) of central memoryT-cell (T_(CM)), thereby producing a plurality of modified centralmemory T-cells (T_(CM)). In certain embodiments, the cell-surfacemarkers comprise one or more of CD45RO, CD95, CCR7, and CD62L. Incertain embodiments of this method, the transposon is a plasmid DNAtransposon with a sequence encoding the antigen receptor or thetherapeutic protein is flanked by two cis-regulatory insulator elements.In certain embodiments, the transposon is a piggyBac transposon. Incertain embodiments, and, in particular, those embodiments wherein thetransposon is a piggyBac transposon, the transposase is a piggyBac™ or aSuper piggyBac™ (SPB) transposase. In certain embodiments of thismethod, the transposon is a Sleeping Beauty transposon. In certainembodiments, and, in particular, those embodiments wherein thetransposon is a Sleeping Beauty transposon, the transposase is aSleeping Beauty transposase or a hyperactive Sleeping Beauty transposase(SB100X). In certain embodiments of this method, the transposon is aHelraiser transposon. In certain embodiments, and, in particular, thoseembodiments wherein the transposon is a Helraiser transposon, thetransposase is a Helitron transposase. In certain embodiments of thismethod, the transposon is a Tol2 transposon. In certain embodiments,including those embodiments wherein the transposon is a Tol2 transposon,the transposase is a Tol2 transposase.

The disclosure provides a method of producing a composition comprising aplurality of modified stem memory T-cells (T_(SCM)) and a plurality ofmodified central memory T-cells (T_(CM)), comprising introducing into aplurality of primary human T cell (a) a transposon compositioncomprising a transposon comprising an antigen receptor or a therapeuticprotein and (b) a transposase composition comprising a transposase or asequence encoding the transposase; to produce a composition comprising aplurality of modified T_(SCM) and a plurality of modified T_(CM),wherein the plurality of modified T_(SCM) expresses one or more CD62L,CD45RA, CD28, CCR7, CD127, CD45RO, CD95, CD95 and IL-2Rβ and theplurality of modified T_(CM) expresses one or more CD45RO, CD95, IL-2Rβ,CCR7, and CD62L, thereby producing a composition comprising a pluralityof modified T_(SCM) and a plurality of modified T_(CM). In certainembodiments of this method, the modified stem memory T-cells (T_(SCM))comprise at least 1%, 2%, 5%, 7%, 10%, 15%, 20%, 25%, 30%, 35%, 40%,45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99% or anypercentage of cells in between of the total number of cells of thecomposition. In certain embodiments of this method, the modified centralmemory T-cells (T_(CM)) comprise at least 1%, 2%, 5%, 7%, 10%, 15%, 20%,25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,95%, 97%, 99% or any percentage of cells in between of the total numberof cells of the composition. In certain embodiments of this method, themodified stem memory T-cells (T_(SCM)) comprise at least 10% of thetotal number of cells of the composition and the modified central memoryT-cells (T_(CM)) comprise at least 90% of the total number of cells ofthe composition. In certain embodiments of this method, the modifiedstem memory T-cells (T_(SCM)) comprise at least 90% of the total numberof cells of the composition and the modified central memory T-cells(T_(CM)) comprise at least 10% of the total number of cells of thecomposition. In certain embodiments of this method, the modified stemmemory T-cells (T_(SCM)) comprise at least 20% of the total number ofcells of the composition and the modified central memory T-cells(T_(CM)) comprise at least 80% of the total number of cells of thecomposition. In certain embodiments of this method, the modified stemmemory T-cells (T_(SCM)) comprise at least 80% of the total number ofcells of the composition and the modified central memory T-cells(T_(CM)) comprise at least 20% of the total number of cells of thecomposition. In certain embodiments of this method, the modified stemmemory T-cells (T_(SCM)) comprise at least 30% of the total number ofcells of the composition and the modified central memory T-cells(T_(CM)) comprise at least 70% of the total number of cells of thecomposition. In certain embodiments of this method, the modified stemmemory T-cells (T_(SCM)) comprise at least 70% of the total number ofcells of the composition and the modified central memory T-cells(T_(CM)) comprise at least 30% of the total number of cells of thecomposition. In certain embodiments of this method, the modified stemmemory T-cells (T_(SCM)) comprise at least 40% of the total number ofcells of the composition and the modified central memory T-cells(T_(CM)) comprise at least 60% of the total number of cells of thecomposition. In certain embodiments of this method, the modified stemmemory T-cells (T_(SCM)) comprise at least 60% of the total number ofcells of the composition and the modified central memory T-cells(T_(CM)) comprise at least 40% of the total number of cells of thecomposition. In certain embodiments of this method, the modified stemmemory T-cells (T_(SCM)) comprise at least 50% of the total number ofcells of the composition and the modified central memory T-cells(T_(CM)) comprise at least 50% of the total number of cells of thecomposition. In certain embodiments of this method, the transposon is aplasmid DNA transposon with a sequence encoding the antigen receptor orthe therapeutic protein is flanked by two cis-regulatory insulatorelements. In certain embodiments, the transposon is a piggyBactransposon. In certain embodiments, and, in particular, thoseembodiments wherein the transposon is a piggyBac transposon, thetransposase is a piggyBac™ or a Super piggyBac™ (SPB) transposase. Incertain embodiments of this method, the transposon is a Sleeping Beautytransposon. In certain embodiments, and, in particular, thoseembodiments wherein the transposon is a Sleeping Beauty transposon, thetransposase is a Sleeping Beauty transposase or a hyperactive SleepingBeauty transposase (SB100X). In certain embodiments of this method, thetransposon is a Helraiser transposon. In certain embodiments, and, inparticular, those embodiments wherein the transposon is a Helraisertransposon, the transposase is a Helitron transposase. In certainembodiments of this method, the transposon is a Tol2 transposon. Incertain embodiments, including those embodiments wherein the transposonis a Tol2 transposon, the transposase is a Tol2 transposase.

In certain embodiments of the methods of the disclosure, the transposonmay be derived or recombined from any species. Alternatively, or inaddition, the transposon may be synthetic.

In certain embodiments of the methods of the disclosure, the antigenreceptor is a T-cell receptor. In certain embodiments, the T-cellreceptor is naturally-occurring. In certain embodiments, the T-cellreceptor is not naturally-occurring. In certain embodiments, and, inparticular, those embodiments wherein the T-cell receptor is notnaturally-occurring, the T-cell receptor comprises one or moremutation(s) compared to a wild-type T-cell receptor. In certainembodiments, and, in particular, those embodiments wherein the T-cellreceptor is not naturally-occurring, the T-cell receptor is arecombinant T-cell receptor. In certain embodiments of this method, theantigen receptor is a Chimeric Antigen Receptor (CAR). In certainembodiments, the CAR is a CARTyrin. In certain embodiments, the CARcomprises one or more VHH sequence(s). In certain embodiments, the CARis a VCAR.

In certain embodiments of the methods of the disclosure, including thosewherein the method comprises introducing into a primary human T cell (a)a transposon composition comprising a transposon comprising an antigenreceptor and (b) a transposase composition comprising a transposase or asequence encoding the transposase, the methods further compriseintroducing into a primary human T cell (c) a second transposoncomposition comprising a transposon comprising a therapeutic protein, toproduce a modified T cell, wherein the modified T cell is capable ofexpressing the therapeutic protein. In certain embodiments, thetherapeutic protein is a secretable protein and the method produces amodified T cell capable of secreting the therapeutic protein. In certainembodiments, the transposase composition of (b) transposes thetransposon of (a) and the transposon of (c). In certain embodiments,this methods further comprises introducing into the primary human T cell(d) a second transposase composition comprising a transposase or asequence encoding the transposase. In certain embodiments, the secondtransposase composition transposes the transposon of (c). In certainembodiments, the transposase composition of (b) transposes thetransposon of (a) and the transposase composition of (d) transposes thetransposon of (c). In certain embodiments of this method, the transposonis a plasmid DNA transposon with a sequence encoding the antigenreceptor or the therapeutic protein flanked by two cis-regulatoryinsulator elements. In certain embodiments, the transposon is a piggyBactransposon. In certain embodiments, and, in particular, thoseembodiments wherein the transposon is a piggyBac transposon, thetransposase is a piggyBac™ or a Super piggyBac™ (SPB) transposase. Incertain embodiments of this method, the transposon is a Sleeping Beautytransposon. In certain embodiments, and, in particular, thoseembodiments wherein the transposon is a Sleeping Beauty transposon, thetransposase is a Sleeping Beauty transposase or a hyperactive SleepingBeauty transposase (SB100X). In certain embodiments of this method, thetransposon is a Helraiser transposon. In certain embodiments, and, inparticular, those embodiments wherein the transposon is a Helraisertransposon, the transposase is a Helitron transposase. In certainembodiments of this method, the transposon is a Tol2 transposon. Incertain embodiments, including those embodiments wherein the transposonis a Tol2 transposon, the transposase is a Tol2 transposase.

The disclosure provides a method of producing a modified stem memory Tcell (T_(SCM)), comprising: (a) introducing into a primary human T cella composition comprising an antigen receptor to produce a modified Tcell, wherein the antigen receptor or the therapeutic protein is notcontained in a transposon, and (b) contacting the modified T-cell and aT-cell activator composition comprising one or more of an anti-human CD3monospecific tetrameric antibody complex, an anti-human CD28monospecific tetrameric antibody complex and an activation supplement toproduce an activated modified T-cell, wherein the activated modifiedT-cell expresses one or more cell-surface marker(s) of a stem memory Tcell (T_(SCM)), thereby producing a modified stem memory T cell(T_(SCM)). The disclosure provides a method of producing a plurality ofmodified stem memory T cells (T_(SCM)), comprising: (a) introducing intoa plurality of primary human T cells a composition comprising an antigenreceptor to produce a plurality of modified T cells, wherein the antigenreceptor or the therapeutic protein is not contained in a transposon,and (b) contacting the plurality of modified T-cells and a T-cellactivator composition comprising one or more of an anti-human CD3monospecific tetrameric antibody complex, an anti-human CD28monospecific tetrameric antibody complex and an activation supplement toproduce a plurality of activated modified T-cells, wherein at least 2%,5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 65%, 70%, 75%,80%, 85%, 90%, 95%, 99% or any percentage in between of the plurality ofactivated modified T cells expresses one or more cell-surface marker(s)of a stem memory T cell (T_(SCM)), thereby producing a plurality ofactivated modified stem memory T cells (T_(SCM)). In certainembodiments, the method produces a plurality of activated modified Tcells, wherein at least 25% of the plurality of activated modified Tcells expresses one or more cell-surface marker(s) of a stem memory Tcell (T_(SCM)), thereby producing a plurality of activated modified stemmemory T cells (T_(SCM)). In certain embodiments, the method produces aplurality of activated modified T cells, wherein at least 50% of theplurality of activated modified T cells expresses one or morecell-surface marker(s) of a stem memory T cell (T_(SCM)), therebyproducing a plurality of activated modified stem memory T cells(T_(SCM)). In certain embodiments, the method produces a plurality ofactivated modified T cells, wherein at least 60% of the plurality ofactivated modified T cells expresses one or more cell-surface marker(s)of a stem memory T cell (T_(SCM)), thereby producing a plurality ofactivated modified stem memory T cells (T_(SCM)). In certainembodiments, the method produces a plurality of activated modified Tcells, wherein at least 75% of the plurality of activated modified Tcells expresses one or more cell-surface marker(s) of a stem memory Tcell (T_(SCM)), thereby producing a plurality of activated modified stemmemory T cells (T_(SCM)). In certain embodiments, the method produces aplurality of activated modified T cells, wherein at least 80% of theplurality of activated modified T cells expresses one or morecell-surface marker(s) of a stem memory T cell (T_(SCM)), therebyproducing a plurality of activated modified stem memory T cells(T_(SCM)). In certain embodiments, the method produces a plurality ofactivated modified T cells, wherein at least 85% of the plurality ofactivated modified T cells expresses one or more cell-surface marker(s)of a stem memory T cell (T_(SCM)), thereby producing a plurality ofactivated modified stem memory T cells (T_(SCM)). In certainembodiments, the method produces a plurality of activated modified Tcells, wherein at least 90% of the plurality of activated modified Tcells expresses one or more cell-surface marker(s) of a stem memory Tcell (T_(SCM)), thereby producing a plurality of activated modified stemmemory T cells (T_(SCM)). In certain embodiments, the method produces aplurality of activated modified T cells, wherein at least 95% of theplurality of activated modified T cells expresses one or morecell-surface marker(s) of a stem memory T cell (T_(SCM)), therebyproducing a plurality of activated modified stem memory T cells(T_(SCM)). In certain embodiments, the cell-surface markers compriseCD62L and CD45RA. In certain embodiments, the cell-surface markers ofthe activated modified T_(SCM) comprise one or more of CD62L, CD45RA,CD28, CCR7, CD127, CD45RO, CD95, CD95 and IL-2Rβ. In certainembodiments, the cell-surface markers of the activated modified T_(SCM)comprise one or more of CD45RA, CD95, IL-2Rβ, CR7, and CD62L.

In certain embodiments of the methods of the disclosure of producing amodified stem memory T cell (T_(SCM)), comprising: (a) introducing intoa primary human T cell a composition comprising an antigen receptor toproduce a modified T cell, wherein the antigen receptor or thetherapeutic protein is not contained in a transposon, and (b) contactingthe modified T-cell and a T-cell activator composition comprising one ormore of an anti-human CD3 monospecific tetrameric antibody complex, ananti-human CD28 monospecific tetrameric antibody complex and anactivation supplement to produce an activated modified T-cell, theT-cell activator composition of (b) further comprises an anti-human CD2monospecific tetrameric antibody complex. In certain embodiments, thismethod further comprises the step of (c) contacting the activatedmodified T-cell and a T-cell expansion composition comprising one ormore of human serum albumin, recombinant human insulin, humantransferrin, 2-Mercaptoethanol, Iscove's MDM, and an expansionsupplement to produce a plurality of expanded modified T-cells, whereinat least 2% of the plurality of expanded modified T-cells expresses oneor more cell-surface marker(s) of a stem memory T cell (T_(SCM)). Incertain embodiments of this method, at least 2%, 5%, 10%, 15%, 20%, 25%,30%, 35%, 40%, 45%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% orany percentage in between of the plurality of expanded modified T-cellsexpresses cell-surface marker(s) of a stem memory T cell (T_(SCM)). Incertain embodiments of this method, at least 60% of the plurality ofexpanded modified T-cells expresses cell-surface marker(s) of a stemmemory T cell (T_(SCM)). In certain embodiments, this method furthercomprises the step of (d) enriching the plurality of expanded modifiedT-cells to produce a composition comprising at least 2%, 5%, 10%, 15%,20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,95%, 99% or any percentage in between of modified T-cells that expresscell-surface marker(s) of a stem memory T cell (T_(SCM)). In certainembodiments, this method further comprises the step of (d) enriching theplurality of expanded modified T-cells to produce a compositioncomprising at least 60% of modified T-cells that express cell-surfacemarker(s) of a stem memory T cell (T_(SCM)). In certain embodiments ofthis method, the enriching step comprises isolating modified T-cellsthat express one or more cell-surface marker(s) of a stem memory T cell(T_(SCM)) from the plurality of enriched modified T-cells. In certainembodiments of this method, the enriching step further comprisescontacting the isolated modified T_(SCM) and a T-cell expansioncomposition comprising one or more of human serum albumin, recombinanthuman insulin, human transferrin, 2-Mercaptoethanol, Iscove's MDM, andan expansion supplement to produce a plurality of expanded enrichedmodified T_(SCM). In certain embodiments of this method, the T-cellexpansion composition further comprises one or more of octanoic acid,nicotinamide, 2,4,7,9-tetramethyl-5-decyn-4,7-diol (TMDD), diisopropyladipate (DIPA), n-butyl-benzenesulfonamide, 1,2-benzenedicarboxylicacid, bis(2-methylpropyl) ester, palmitic acid, linoleic acid, oleicacid, stearic acid hydrazide, oleamide, a sterol and an alkane. Incertain embodiments of this method, the T-cell expansion compositionfurther comprises one or more of octanoic acid, palmitic acid, linoleicacid, oleic acid and a sterol. In certain embodiments of this method,the T-cell expansion composition further comprises one or more ofoctanoic acid at a concentration of between 0.9 mg/kg to 90 mg/kg,inclusive of the endpoints; palmitic acid at a concentration of between0.2 mg/kg to 20 mg/kg, inclusive of the endpoints; linoleic acid at aconcentration of between 0.2 mg/kg to 20 mg/kg, inclusive of theendpoints; oleic acid at a concentration of 0.2 mg/kg to 20 mg/kg,inclusive of the endpoints; and a sterol at a concentration of about 0.1mg/kg to 10 mg/kg, inclusive of the endpoints. In certain embodiments ofthis method, the T-cell expansion composition further comprises one ormore of octanoic acid at a concentration of about 9 mg/kg, palmitic acidat a concentration of about 2 mg/kg, linoleic acid at a concentration ofabout 2 mg/kg, oleic acid at a concentration of about 2 mg/kg and asterol at a concentration of about 1 mg/kg. In certain embodiments ofthis method, the T-cell expansion composition further comprises one ormore of octanoic acid at a concentration of between 6.4 μmol/kg and 640μmol/kg, inclusive of the endpoints; palmitic acid at a concentration ofbetween 0.7 μmol/kg and 70 μmol/kg, inclusive of the endpoints; linoleicacid at a concentration of between 0.75 μmol/kg and 75 μmol/kg,inclusive of the endpoints; oleic acid at a concentration of between0.75 μmol/kg and 75 μmol/kg, inclusive of the endpoints; and a sterol ata concentration of between 0.25 μmol/kg and 25 μmol/kg, inclusive of theendpoints. In certain embodiments of this method, the T-cell expansioncomposition further comprises one or more of octanoic acid at aconcentration of about 64 μmol/kg, palmitic acid at a concentration ofabout 7 μmol/kg, linoleic acid at a concentration of about 7.5 μmol/kg,oleic acid at a concentration of about 7.5 μmol/kg and a sterol at aconcentration of about 2.5 μmol/kg.

The disclosure provides a method of producing a modified central memoryT-cell (T_(CM)), comprising: (a) introducing into a primary human T cella composition comprising an antigen receptor to produce a modified Tcell, wherein the antigen receptor or the therapeutic protein is notcontained in a transposon, and (b) contacting the modified T-cell and aT-cell activator composition comprising one or more of an anti-human CD3monospecific tetrameric antibody complex, an anti-human CD28monospecific tetrameric antibody complex and an activation supplement toproduce an activated modified T-cell, wherein the activated modifiedT-cell expresses one or more cell-surface marker(s) of a central memoryT-cell (T_(CM)), thereby producing a central memory T-cell (T_(CM)). Thedisclosure provides a method of producing a plurality of modifiedcentral memory T-cell (T_(CM)), comprising: (a) introducing into aplurality of primary human T cells a composition comprising an antigenreceptor to produce a plurality of modified T cells, wherein the antigenreceptor or the therapeutic protein is not contained in a transposon,and (b) contacting the plurality of modified T-cells and a T-cellactivator composition comprising one or more of an anti-human CD3monospecific tetrameric antibody complex, an anti-human CD28monospecific tetrameric antibody complex and an activation supplement toproduce a plurality of activated modified T-cells, wherein at least 2%,5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 65%, 70%, 75%,80%, 85%, 90%, 95%, 99% or any percentage in between of the plurality ofactivated modified T cells expresses one or more cell-surface marker(s)of a central memory T-cell (T_(CM)), thereby producing a plurality ofactivated modified central memory T-cell (T_(CM)). In certainembodiments, the method produces a plurality of activated modified Tcells, wherein at least 25% of the plurality of activated modified Tcells expresses one or more cell-surface marker(s) of a central memory Tcell (T_(CM)), thereby producing a plurality of activated modifiedcentral memory T cell (T_(CM)). In certain embodiments, the methodproduces a plurality of activated modified T cells, wherein at least 50%of the plurality of activated modified T cells expresses one or morecell-surface marker(s) of a central memory T cell (T_(CM)), therebyproducing a plurality of activated modified central memory T cell(T_(CM)). In certain embodiments, the method produces a plurality ofactivated modified T cells, wherein at least 60% of the plurality ofactivated modified T cells expresses one or more cell-surface marker(s)of a central memory T cell (T_(CM)), thereby producing a plurality ofactivated modified central memory T cell (T_(CM)). In certainembodiments, the method produces a plurality of activated modified Tcells, wherein at least 75% of the plurality of activated modified Tcells expresses one or more cell-surface marker(s) of a central memory Tcell (T_(CM)), thereby producing a plurality of activated modifiedcentral memory T cell (T_(CM)). In certain embodiments, the methodproduces a plurality of activated modified T cells, wherein at least 80%of the plurality of activated modified T cells expresses one or morecell-surface marker(s) of a central memory T cell (T_(CM)), therebyproducing a plurality of activated modified central memory T cell(T_(CM)). In certain embodiments, the method produces a plurality ofactivated modified T cells, wherein at least 85% of the plurality ofactivated modified T cells expresses one or more cell-surface marker(s)of a central memory T cell (T_(CM)), thereby producing a plurality ofactivated modified central memory T cell (T_(CM)). In certainembodiments, the method produces a plurality of activated modified Tcells, wherein at least 90% of the plurality of activated modified Tcells expresses one or more cell-surface marker(s) of a central memory Tcell (T_(CM)), thereby producing a plurality of activated modifiedcentral memory T cell (T_(CM)). In certain embodiments, the methodproduces a plurality of activated modified T cells, wherein at least 95%of the plurality of activated modified T cells expresses one or morecell-surface marker(s) of a central memory T cell (T_(CM)), therebyproducing a plurality of activated modified central memory T cell(T_(CM)). In certain embodiments, the cell-surface markers of theactivated modified T_(CM) comprise one or more of CD45RO, CD95, IL-2Rβ,CCR7, and CD62L.

In certain embodiments of the methods of the disclosure of producing amodified central memory T cell (T_(CM)), comprising: (a) introducinginto a primary human T cell a composition comprising an antigen receptorto produce a modified T cell, wherein the antigen receptor or thetherapeutic protein is not contained in a transposon, and (b) contactingthe modified T-cell and a T-cell activator composition comprising one ormore of an anti-human CD3 monospecific tetrameric antibody complex, ananti-human CD28 monospecific tetrameric antibody complex and anactivation supplement to produce an activated modified T-cell, theT-cell activator composition of (b) further comprises an anti-human CD2monospecific tetrameric antibody complex. In certain embodiments, thismethod further comprises the step of (c) contacting the activatedmodified T-cell and a T-cell expansion composition comprising one ormore of human serum albumin, recombinant human insulin, humantransferrin, 2-Mercaptoethanol, Iscove's MDM, and an expansionsupplement to produce a plurality of expanded modified T-cells, whereinat least 2% of the plurality of expanded modified T-cells expresses oneor more cell-surface marker(s) of a central memory T cell (T_(CM)). Incertain embodiments of this method, at least 2%, 5%, 10%, 15%, 20%, 25%,30%, 35%, 40%, 45%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% orany percentage in between of the plurality of expanded modified T-cellsexpresses cell-surface marker(s) of a central memory T cell (T_(CM)). Incertain embodiments of this method, at least 60% of the plurality ofexpanded modified T-cells expresses cell-surface marker(s) of a centralmemory T cell (T_(CM)). In certain embodiments, this method furthercomprises the step of (d) enriching the plurality of expanded modifiedT-cells to produce a composition comprising at least 2%, 5%, 10%, 15%,20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,95%, 99% or any percentage in between of modified T-cells that expresscell-surface marker(s) of a central memory T cell (T_(CM)). In certainembodiments, this method further comprises the step of (d) enriching theplurality of expanded modified T-cells to produce a compositioncomprising at least 60% of modified T-cells that express cell-surfacemarker(s) of a central memory T cell (T_(CM)). In certain embodiments ofthis method, the enriching step comprises isolating modified T-cellsthat express one or more cell-surface marker(s) of a central memory Tcell (T_(CM)) from the plurality of enriched modified T-cells. Incertain embodiments of this method, the enriching step further comprisescontacting the isolated modified T_(CM) and a T-cell expansioncomposition comprising one or more of human serum albumin, recombinanthuman insulin, human transferrin, 2-Mercaptoethanol, Iscove's MDM, andan expansion supplement to produce a plurality of expanded enrichedmodified T_(CM). In certain embodiments of this method, the T-cellexpansion composition further comprises one or more of octanoic acid,nicotinamide, 2,4,7,9-tetramethyl-5-decyn-4,7-diol (TMDD), diisopropyladipate (DIPA), n-butyl-benzenesulfonamide, 1,2-benzenedicarboxylicacid, bis(2-methylpropyl) ester, palmitic acid, linoleic acid, oleicacid, stearic acid hydrazide, oleamide, a sterol and an alkane. Incertain embodiments of this method, the T-cell expansion compositionfurther comprises one or more of octanoic acid, palmitic acid, linoleicacid, oleic acid and a sterol. In certain embodiments of this method,the T-cell expansion composition further comprises one or more ofoctanoic acid at a concentration of between 0.9 mg/kg to 90 mg/kg,inclusive of the endpoints; palmitic acid at a concentration of between0.2 mg/kg to 20 mg/kg, inclusive of the endpoints; linoleic acid at aconcentration of between 0.2 mg/kg to 20 mg/kg, inclusive of theendpoints; oleic acid at a concentration of 0.2 mg/kg to 20 mg/kg,inclusive of the endpoints; and a sterol at a concentration of about 0.1mg/kg to 10 mg/kg, inclusive of the endpoints. In certain embodiments ofthis method, the T-cell expansion composition further comprises one ormore of octanoic acid at a concentration of about 9 mg/kg, palmitic acidat a concentration of about 2 mg/kg, linoleic acid at a concentration ofabout 2 mg/kg, oleic acid at a concentration of about 2 mg/kg and asterol at a concentration of about 1 mg/kg. In certain embodiments ofthis method, the T-cell expansion composition further comprises one ormore of octanoic acid at a concentration of between 6.4 μmol/kg and 640μmol/kg, inclusive of the endpoints; palmitic acid at a concentration ofbetween 0.7 μmol/kg and 70 μmol/kg, inclusive of the endpoints; linoleicacid at a concentration of between 0.75 μmol/kg and 75 μmol/kg,inclusive of the endpoints; oleic acid at a concentration of between0.75 μmol/kg and 75 μmol/kg, inclusive of the endpoints; and a sterol ata concentration of between 0.25 μmol/kg and 25 μmol/kg, inclusive of theendpoints. In certain embodiments of this method, the T-cell expansioncomposition further comprises one or more of octanoic acid at aconcentration of about 64 μmol/kg, palmitic acid at a concentration ofabout 7 μmol/kg, linoleic acid at a concentration of about 7.5 μmol/kg,oleic acid at a concentration of about 7.5 μmol/kg and a sterol at aconcentration of about 2.5 μmol/kg.

The disclosure provides a method of producing a composition comprising aplurality of modified stem memory T-cells (T_(SCM)) and a plurality ofmodified central memory T-cells (T_(CM)), comprising: (a) introducinginto a plurality of primary human T cells a composition comprising anantigen receptor to produce a plurality of modified T cells, wherein theantigen receptor or the therapeutic protein is not contained in atransposon, and (b) contacting the plurality of modified T-cells and aT-cell activator composition comprising one or more of an anti-human CD3monospecific tetrameric antibody complex, an anti-human CD28monospecific tetrameric antibody complex and an activation supplement toproduce a composition comprising a plurality of activated modified stemmemory T-cells (T_(SCM)) and a plurality of activated modified centralmemory T-cells (T_(CM)), wherein the plurality of activated modifiedT_(SCM) expresses one or more CD62L, CD45RA, CD28, CCR7, CD127, CD45RO,CD95, CD95 and IL-2Rβ and the plurality of activated modified T_(CM)expresses one or more CD45RO, CD95, IL-2Rβ, CCR7, and CD62L, therebyproducing a composition comprising a plurality of modified T_(SCM) and aplurality of modified T_(CM). In certain embodiments of this method, themodified stem memory T-cells (T_(SCM)) comprise at least 1%, 2%, 5%, 7%,10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, 95%, 97%, 99% or any percentage of cells in between ofthe total number of cells of the composition. In certain embodiments ofthis method, the modified central memory T-cells (T_(CM)) comprise atleast 1%, 2%, 5%, 7%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%,60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99% or any percentage ofcells in between of the total number of cells of the composition. Incertain embodiments of this method, the modified stem memory T-cells(T_(SCM)) comprise at least 10% of the total number of cells of thecomposition and the modified central memory T-cells (T_(CM)) comprise atleast 90% of the total number of cells of the composition. In certainembodiments of this method, the modified stem memory T-cells (T_(SCM))comprise at least 90% of the total number of cells of the compositionand the modified central memory T-cells (T_(CM)) comprise at least 10%of the total number of cells of the composition. In certain embodimentsof this method, the modified stem memory T-cells (T_(SCM)) comprise atleast 20% of the total number of cells of the composition and themodified central memory T-cells (T_(CM)) comprise at least 80% of thetotal number of cells of the composition. In certain embodiments of thismethod, the modified stem memory T-cells (T_(SCM)) comprise at least 80%of the total number of cells of the composition and the modified centralmemory T-cells (T_(CM)) comprise at least 20% of the total number ofcells of the composition. In certain embodiments of this method, themodified stem memory T-cells (T_(SCM)) comprise at least 30% of thetotal number of cells of the composition and the modified central memoryT-cells (T_(CM)) comprise at least 70% of the total number of cells ofthe composition. In certain embodiments of this method, the modifiedstem memory T-cells (T_(SCM)) comprise at least 70% of the total numberof cells of the composition and the modified central memory T-cells(T_(CM)) comprise at least 30% of the total number of cells of thecomposition. In certain embodiments of this method, the modified stemmemory T-cells (T_(SCM)) comprise at least 40% of the total number ofcells of the composition and the modified central memory T-cells(T_(CM)) comprise at least 60% of the total number of cells of thecomposition. In certain embodiments of this method, the modified stemmemory T-cells (T_(SCM)) comprise at least 60% of the total number ofcells of the composition and the modified central memory T-cells(T_(CM)) comprise at least 40% of the total number of cells of thecomposition. In certain embodiments of this method, the modified stemmemory T-cells (T_(SCM)) comprise at least 50% of the total number ofcells of the composition and the modified central memory T-cells(T_(CM)) comprise at least 50% of the total number of cells of thecomposition.

In certain embodiments of methods of the disclosure of producing acomposition comprising a plurality of modified stem memory T-cells(T_(SCM)) and a plurality of modified central memory T-cells (T_(CM)),comprising: (a) introducing into a plurality of primary human T cells acomposition comprising an antigen receptor to produce a plurality ofmodified T cells, wherein the antigen receptor or the therapeuticprotein is not contained in a transposon, and (b) contacting theplurality of modified T-cells and a T-cell activator compositioncomprising one or more of an anti-human CD3 monospecific tetramericantibody complex, an anti-human CD28 monospecific tetrameric antibodycomplex and an activation supplement to produce a composition comprisinga plurality of activated modified stem memory T-cells (T_(SCM)) and aplurality of activated modified central memory T-cells (T_(CM)), theT-cell activator composition of (b) further comprises an anti-human CD2monospecific tetrameric antibody complex. In certain embodiments, thismethod further comprises the step of (c) contacting the composition theplurality of activated modified stem memory T-cells (T_(SCM)) and theplurality of activated modified central memory T-cells (T_(CM)) with aT-cell expansion composition comprising one or more of human serumalbumin, recombinant human insulin, human transferrin,2-Mercaptoethanol, Iscove's MDM, and an expansion supplement to producea plurality of expanded modified T-cells, wherein the plurality ofexpanded modified T_(SCM) expresses one or more CD62L, CD45RA, CD28,CCR7, CD127, CD45RO, CD95, CD95 and IL-2Rβ and the plurality of expandedmodified T_(CM) expresses one or more CD45RO, CD95, IL-2Rβ, CCR7, andCD62L, thereby producing a composition comprising a plurality ofexpanded modified T_(SCM) and a plurality of expanded modified T_(CM).In certain embodiments of this method, the enriching step comprisesisolating modified T-cells that express one or more cell-surfacemarker(s) of a stem memory T cell (T_(SCM)) from the plurality ofenriched modified T-cells or isolating modified T-cells that express oneor more cell-surface marker(s) of a central memory T cell (T_(CM)) fromthe plurality of enriched modified T-cells. In certain embodiments ofthis method, the enriching step comprises isolating modified T-cellsthat express one or more cell-surface marker(s) of a stem memory T cell(T_(SCM)) from the plurality of enriched modified T-cells and isolatingmodified T-cells that express one or more cell-surface marker(s) of acentral memory T cell (T_(CM)) from the plurality of enriched modifiedT-cells. In certain embodiments of this method, the enriching stepfurther comprises contacting the composition comprising the isolatedmodified T_(SCM) and the isolated modified T_(CM) with a T-cellexpansion composition comprising one or more of human serum albumin,recombinant human insulin, human transferrin, 2-Mercaptoethanol,Iscove's MDM, and an expansion supplement to produce a compositioncomprising a plurality of expanded enriched modified T_(SCM) and aplurality of expanded enriched modified T_(CM). In certain embodimentsof this method, the T-cell expansion composition further comprises oneor more of octanoic acid, nicotinamide,2,4,7,9-tetramethyl-5-decyn-4,7-diol (TMDD), diisopropyl adipate (DIPA),n-butyl-benzenesulfonamide, 1,2-benzenedicarboxylic acid,bis(2-methylpropyl) ester, palmitic acid, linoleic acid, oleic acid,stearic acid hydrazide, oleamide, a sterol and an alkane. In certainembodiments of this method, the T-cell expansion composition furthercomprises one or more of octanoic acid, palmitic acid, linoleic acid,oleic acid and a sterol. In certain embodiments of this method, theT-cell expansion composition further comprises one or more of octanoicacid at a concentration of between 0.9 mg/kg to 90 mg/kg, inclusive ofthe endpoints; palmitic acid at a concentration of between 0.2 mg/kg to20 mg/kg, inclusive of the endpoints; linoleic acid at a concentrationof between 0.2 mg/kg to 20 mg/kg, inclusive of the endpoints; oleic acidat a concentration of 0.2 mg/kg to 20 mg/kg, inclusive of the endpoints;and a sterol at a concentration of about 0.1 mg/kg to 10 mg/kg,inclusive of the endpoints. In certain embodiments of this method, theT-cell expansion composition further comprises one or more of octanoicacid at a concentration of about 9 mg/kg, palmitic acid at aconcentration of about 2 mg/kg, linoleic acid at a concentration ofabout 2 mg/kg, oleic acid at a concentration of about 2 mg/kg and asterol at a concentration of about 1 mg/kg. In certain embodiments ofthis method, the T-cell expansion composition further comprises one ormore of octanoic acid at a concentration of between 6.4 μmol/kg and 640μmol/kg, inclusive of the endpoints; palmitic acid at a concentration ofbetween 0.7 μmol/kg and 70 μmol/kg, inclusive of the endpoints; linoleicacid at a concentration of between 0.75 μmol/kg and 75 μmol/kg,inclusive of the endpoints; oleic acid at a concentration of between0.75 μmol/kg and 75 μmol/kg, inclusive of the endpoints; and a sterol ata concentration of between 0.25 μmol/kg and 25 μmol/kg, inclusive of theendpoints. In certain embodiments of this method, the T-cell expansioncomposition further comprises one or more of octanoic acid at aconcentration of about 64 μmol/kg, palmitic acid at a concentration ofabout 7 μmol/kg, linoleic acid at a concentration of about 7.5 μmol/kg,oleic acid at a concentration of about 7.5 μmol/kg and a sterol at aconcentration of about 2.5 μmol/kg. In certain embodiments of thismethod, the modified stem memory T-cells (T_(SCM)) comprise at least 1%,2%, 5%, 7%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, 95%, 97%, 99% or any percentage of cells inbetween of the total number of cells of the composition. In certainembodiments of this method, the modified central memory T-cells (T_(CM))comprise at least 1%, 2%, 5%, 7%, 10%, 15%, 20%, 25%, 30%, 35%, 40%,45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99% or anypercentage of cells in between of the total number of cells of thecomposition. In certain embodiments of this method, the modified stemmemory T-cells (T_(SCM)) comprise at least 10% of the total number ofcells of the composition and the modified central memory T-cells(T_(CM)) comprise at least 90% of the total number of cells of thecomposition. In certain embodiments of this method, the modified stemmemory T-cells (T_(SCM)) comprise at least 90% of the total number ofcells of the composition and the modified central memory T-cells(T_(CM)) comprise at least 10% of the total number of cells of thecomposition. In certain embodiments of this method, the modified stemmemory T-cells (T_(SCM)) comprise at least 20% of the total number ofcells of the composition and the modified central memory T-cells(T_(CM)) comprise at least 80% of the total number of cells of thecomposition. In certain embodiments of this method, the modified stemmemory T-cells (T_(SCM)) comprise at least 80% of the total number ofcells of the composition and the modified central memory T-cells(T_(CM)) comprise at least 20% of the total number of cells of thecomposition. In certain embodiments of this method, the modified stemmemory T-cells (T_(SCM)) comprise at least 30% of the total number ofcells of the composition and the modified central memory T-cells(T_(CM)) comprise at least 70% of the total number of cells of thecomposition. In certain embodiments of this method, the modified stemmemory T-cells (T_(SCM)) comprise at least 70% of the total number ofcells of the composition and the modified central memory T-cells(T_(CM)) comprise at least 30% of the total number of cells of thecomposition. In certain embodiments of this method, the modified stemmemory T-cells (T_(SCM)) comprise at least 40% of the total number ofcells of the composition and the modified central memory T-cells(T_(CM)) comprise at least 60% of the total number of cells of thecomposition. In certain embodiments of this method, the modified stemmemory T-cells (T_(SCM)) comprise at least 60% of the total number ofcells of the composition and the modified central memory T-cells(T_(CM)) comprise at least 40% of the total number of cells of thecomposition. In certain embodiments of this method, the modified stemmemory T-cells (T_(SCM)) comprise at least 50% of the total number ofcells of the composition and the modified central memory T-cells(T_(CM)) comprise at least 50% of the total number of cells of thecomposition.

In certain embodiments of the methods of producing an activated modifiedT_(SCM) or T_(CM) of the disclosure, including those methods comprising(a) introducing into a plurality of primary human T cells a compositioncomprising an antigen receptor to produce a plurality of modified Tcells, wherein the antigen receptor or the therapeutic protein is notcontained in a transposon, and (b) contacting the plurality of modifiedT-cells and a T-cell activator composition comprising one or more of ananti-human CD3 monospecific tetrameric antibody complex, the introducingstep comprises a homologous recombination. In certain embodiments of theintroduction step comprising a homologous recombination, a genomicediting composition contacts a genomic sequence of at least one primaryT cell of the plurality of T cells. In certain embodiments of theintroduction step comprising a homologous recombination, a genomicediting composition contacts a genomic sequence of a portion of primaryT cells of the plurality of T cells. In certain embodiments, the portionof primary T cells is at least 1%, 2%, 5%, 7%, 10%, 15%, 20%, 25%, 30%,35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%,99% or any percentage in between of the total number of primary T cellsin the plurality of T cells. In certain embodiments of the introductionstep comprising a homologous recombination, a genomic editingcomposition contacts a genomic sequence of each primary T cell of theplurality of T cells. In certain embodiments of the introduction stepcomprising a homologous recombination, a genomic editing compositioninduces a single strand break. In certain embodiments of theintroduction step comprising a homologous recombination, a genomicediting composition induces a double strand break. In certainembodiments of the introduction step comprising a homologousrecombination, the introduction step further comprises a donor sequencecomposition. In certain embodiments, the donor sequence compositioncomprises a sequence encoding the antigen receptor. In certainembodiments, the donor sequence composition comprises a sequenceencoding the antigen receptor, a 5′ genomic sequence and a 3′ genomicsequence, wherein the 5′ genomic sequence is homologous or identical toa genomic sequence of the primary T cell that is 5′ to the break pointinduced by the genomic editing composition and the 3′ genomic sequenceis homologous or identical to a genomic sequence of the primary T cellthat is 3′ to the break point induced by the genomic editingcomposition. In certain embodiments of the introduction step comprisinga homologous recombination, the genomic editing composition and donorsequence composition are contacted with the genomic sequencesimultaneously or sequentially. In certain embodiments of theintroduction step comprising a homologous recombination, the genomicediting composition and donor sequence composition are contacted withthe genomic sequence sequentially, and the genomic editing compositionis provided first. In certain embodiments of the introduction stepcomprising a homologous recombination, the genomic editing compositioncomprises a sequence encoding a DNA binding domain and a sequenceencoding a nuclease domain. In certain embodiments of the introductionstep comprising a homologous recombination, the genomic editingcomposition comprises a DNA binding domain and a nuclease domain. Incertain embodiments of the genomic editing composition, the DNA bindingdomain comprises a guide RNA (gRNA). In certain embodiments of thegenomic editing composition, the DNA binding domain comprises aDNA-binding domain of a TALEN. In certain embodiments of the genomicediting composition, the DNA binding domain comprises a DNA-bindingdomain of a ZFN. In certain embodiments of the genomic editingcomposition, the nuclease domain comprises a Cas9 nuclease or a sequencethereof. In certain embodiments of the genomic editing composition, thenuclease domain comprises an inactive Cas9 (SEQ ID NO: 33, comprising asubstitution of a Alanine (A) for Aspartic Acid (D) at position 10(D10A) and a substitution of Alanine (A) for Histidine (H) at position840 (H840A)). In certain embodiments of the genomic editing composition,the nuclease domain comprises a short and inactive Cas9 (SEQ ID NO: 32,comprising a substitution of an Alanine (A) for an Aspartic Acid (D) atposition 10 (D10A) and a substitution of an Alanine (A) for anAsparagine (N) at position 540 (N540A)). In certain embodiments of thegenomic editing composition, the nuclease domain comprises or furthercomprises a type IIS endonuclease. In certain embodiments of the genomicediting composition, the type IIS endonuclease comprises AciI, Mn1I,AlwI, BbvI, BccI, BceAI, BsmAI, BsmFI, BspCNI, BsrI, BtsCI, HgaI, HphI,HpyAV, Mbo1I, My1I, PleI, SfaNI, AcuI, BciVI, BfuAI, BmgBI, BmrI, BpmI,BpuEI, BsaI, BseRI, BsgI, BsmI, BspMI, BsrBI, BsrBI, BsrDI, BtgZI, BtsI,EarI, EciI, MmeI, NmeAIII, BbvCI, Bpu10I, BspQI, SapI, BaeI, BsaXI,CspCI, BfiI, MboII, Acc36I, FokI or Clo051. In certain embodiments, thetype IIS endonuclease comprises Clo051. In certain embodiments of thegenomic editing composition, the nuclease domain comprises or furthercomprises a TALEN or a nuclease domain thereof. In certain embodimentsof the genomic editing composition, the nuclease domain comprises orfurther comprises a ZFN or a nuclease domain thereof. In certainembodiments of the introduction step comprising a homologousrecombination, the genomic editing composition induces a break in agenomic sequence and the donor sequence composition is inserted usingthe endogenous DNA repair mechanisms of the primary T cell. In certainembodiments of the introduction step comprising a homologousrecombination, the insertion of the donor sequence compositioneliminates a DNA binding site of the genomic editing composition,thereby preventing further activity of the genomic editing composition.

In certain embodiments of the methods of producing an activated modifiedT_(SCM) or T_(CM) of the disclosure, including those methods comprising(a) introducing into a plurality of primary human T cells a compositioncomprising an antigen receptor to produce a plurality of modified Tcells, wherein the antigen receptor or the therapeutic protein is notcontained in a transposon, and (b) contacting the plurality of modifiedT-cells and a T-cell activator composition comprising one or more of ananti-human CD3 monospecific tetrameric antibody complex, an anti-humanCD28 monospecific tetrameric antibody complex and an activationsupplement, a viral vector comprises the antigen receptor. In certainembodiments, the viral vector comprises one or more sequences isolated,derived, or recombined from an RNA virus. In certain embodiments, theRNA virus is a single-stranded or a double-stranded virus. In certainembodiments, the viral vector comprises one or more sequences isolated,derived, or recombined from a DNA virus. In certain embodiments, the DNAvirus is a single-stranded or a double-stranded virus. In certainembodiments, the virus is replication-defective.

In certain embodiments of the methods of producing an activated modifiedT_(SCM) or T_(CM) of the disclosure, including those methods comprising(a) introducing into a plurality of primary human T cells a compositioncomprising an antigen receptor to produce a plurality of modified Tcells, wherein the antigen receptor or the therapeutic protein is notcontained in a transposon, and (b) contacting the plurality of modifiedT-cells and a T-cell activator composition comprising one or more of ananti-human CD3 monospecific tetrameric antibody complex, an anti-humanCD28 monospecific tetrameric antibody complex and an activationsupplement, a viral vector comprises the antigen receptor. In certainembodiments, the viral vector comprises a sequence isolated or derivedfrom a retrovirus. In certain embodiments, the viral vector comprises asequence isolated or derived from a lentivirus.

In certain embodiments of the methods of producing an activated modifiedT_(SCM) or T_(CM) of the disclosure, including those methods comprising(a) introducing into a plurality of primary human T cells a compositioncomprising an antigen receptor to produce a plurality of modified Tcells, wherein the antigen receptor or the therapeutic protein is notcontained in a transposon, and (b) contacting the plurality of modifiedT-cells and a T-cell activator composition comprising one or more of ananti-human CD3 monospecific tetrameric antibody complex, an anti-humanCD28 monospecific tetrameric antibody complex and an activationsupplement, a viral vector comprises the antigen receptor. In certainembodiments, the viral vector comprises a sequence isolated or derivedfrom a retrovirus. In certain embodiments, the viral vector comprises asequence isolated or derived from a gamma retrovirus.

In certain embodiments of the methods of producing an activated modifiedT_(SCM) or T_(CM) of the disclosure, including those methods comprising(a) introducing into a plurality of primary human T cells a compositioncomprising an antigen receptor to produce a plurality of modified Tcells, wherein the antigen receptor or the therapeutic protein is notcontained in a transposon, and (b) contacting the plurality of modifiedT-cells and a T-cell activator composition comprising one or more of ananti-human CD3 monospecific tetrameric antibody complex, an anti-humanCD28 monospecific tetrameric antibody complex and an activationsupplement, a viral vector comprises the antigen receptor. In certainembodiments, the viral vector comprises a sequence isolated or derivedfrom an adeno-associated virus (AAV). In certain embodiments, the AAV isa serotype AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10or AAV11. In certain embodiments, the AAV comprises a sequence from oneor more of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10or AAV11. In certain embodiments, the AAV comprises a sequence isolated,derived, or recombined from one or more of AAV1, AAV2, AAV3, AAV4, AAV5,AAV6, AAV7, AAV8, AAV9, AAV10 or AAV11. In certain embodiments, the AAVcomprises a sequence isolated, derived, or recombined from AAV2. Incertain embodiments, including those in which the vector crosses theblood brain barrier (BBB), the AAV comprises a sequence isolated,derived, or recombined from AAV9. Exemplary adeno-associated viruses andrecombinant adeno-associated viruses of the disclosure include, but arenot limited to, self-complementary AAV (scAAV) and AAV hybridscontaining the genome of one serotype and the capsid of another serotype(e.g. AAV2/5, AAV-DJ and AAV-DJ8). Exemplary adeno-associated virusesand recombinant adeno-associated viruses of the disclosure include, butare not limited to, rAAV-LK03, rAAV-NP59 and rAAV-NP84.

In certain embodiments of the methods of producing an activated modifiedT_(SCM) or T_(CM) of the disclosure, a nucleic acid vector comprises theantigen receptor. In certain embodiments, a DNA vector comprises theantigen receptor. In certain embodiments, an mRNA vector comprises theantigen receptor. In certain embodiments, the nucleic acid vector is aplasmid or a minicircle vector.

In certain embodiments of the methods of producing an activated modifiedT_(SCM) or T_(CM) of the disclosure, a nanoparticle vector comprises theantigen receptor. Nanoparticles may be comprised of polymers disclosedin, for example, International Patent Publication No. WO 2012/094679,International Patent Publication No. WO 2016/022805, InternationalPatent Publication No. WO/2011/133635, International Patent PublicationNo. WO/2016/090111, International Patent Publication No. WO/2017/004498,WO/2017/004509, International Patent Application No. PCT/US2017/030271,U.S. Pat. Nos. 6,835,394, 7,217,427, and 7,867,512.

In certain embodiments of the methods of producing an activated modifiedT_(SCM) or T_(CM) of the disclosure, the antigen receptor is a T-cellreceptor. In certain embodiments, the T-cell receptor isnaturally-occurring. In certain embodiments, the T-cell receptor is notnaturally-occurring. In certain embodiments, and, in particular, thoseembodiments wherein the T-cell receptor is not naturally-occurring, theT-cell receptor comprises one or more mutation(s) compared to awild-type T-cell receptor. In certain embodiments, and, in particular,those embodiments wherein the T-cell receptor is notnaturally-occurring, the T-cell receptor is a recombinant T-cellreceptor. In certain embodiments of this method, the antigen receptor isa Chimeric Antigen Receptor (CAR). In certain embodiments, the CAR is aCARTyrin. In certain embodiments, the CAR comprises one or more VHHsequence(s). In certain embodiments, the CAR is a VCAR.

In certain embodiments of the methods of producing an activated modifiedT_(SCM) or T_(CM) of the disclosure, including those methods comprising(a) introducing into a plurality of primary human T cells a compositioncomprising an antigen receptor to produce a plurality of modified Tcells, wherein the antigen receptor or the therapeutic protein is notcontained in a transposon, and (b) contacting the plurality of modifiedT-cells and a T-cell activator composition comprising one or more of ananti-human CD3 monospecific tetrameric antibody complex, an anti-humanCD28 monospecific tetrameric antibody complex and an activationsupplement, the method further comprises introducing into the primaryhuman T cell, a composition comprising a therapeutic protein to producea modified T cell capable of expressing the therapeutic protein. Incertain embodiments, the therapeutic protein is a secretable protein andthe method produces a modified T cell capable of secreting thetherapeutic protein. In certain embodiments, the introducing stepcomprises a homologous recombination and a donor sequence comprises asequence encoding the therapeutic protein. In certain embodiments, thedonor sequence that comprises the antigen receptor further comprises thetherapeutic protein. In certain embodiments, a first donor sequencecomprises the antigen receptor and a second donor sequence comprises thetherapeutic protein. In certain embodiments, a vector comprises asequence encoding the therapeutic protein. In certain embodiments, thevector is a viral vector. In certain embodiments, the vector is ananoparticle. In certain embodiments, the vector that comprises theantigen receptor further comprises the therapeutic protein. In certainembodiments, a first vector comprises the antigen receptor and a secondvector template comprises the therapeutic protein.

The disclosure provides a method of producing a modified stem memory Tcell (T_(SCM)), comprising: (a) introducing into a primary human T cella composition comprising an antigen receptor to produce a modified Tcell, wherein a transposon comprises the antigen receptor, and (b)contacting the modified T cell and a T-cell activator compositioncomprising one or more of an anti-human CD3 monospecific tetramericantibody complex, an anti-human CD28 monospecific tetrameric antibodycomplex and an activation supplement to produce an activated modifiedT-cell, wherein the activated modified-T cell expresses one or morecell-surface marker(s) of a stem memory T cell (T_(SCM)), therebyproducing a modified stem memory T cell (T_(SCM)). The disclosureprovides a method of producing a plurality of modified stem memory Tcells (T_(SCM)), comprising: (a) introducing into a plurality of primaryhuman T cells a composition comprising an antigen receptor to produce aplurality of modified T cells, wherein a transposon comprises theantigen receptor, and (b) contacting the plurality of modified T cellsand a T-cell activator composition comprising one or more of ananti-human CD3 monospecific tetrameric antibody complex, an anti-humanCD28 monospecific tetrameric antibody complex and an activationsupplement to produce a plurality of activated modified T-cells, whereinat least 25%, 50%, 60%, 75%, 80%, 85%, 90%, 95% or 99% of the pluralityof activated modified −T cells expresses one or more cell-surfacemarker(s) of a stem memory T cell (T_(SCM)), thereby producing amodified stem memory T cell (T_(SCM)). In certain embodiments of thismethod, at least 60% of the plurality of activated modified −T cellsexpresses one or more cell-surface marker(s) of a stem memory T cell(T_(SCM)). In certain embodiments of this method, the T-cell activatorcomposition of (b) further comprises an anti-human CD2 monospecifictetrameric antibody complex. The disclosure provides a method ofproducing a modified stem memory T cell (T_(SCM)), comprising: (a)introducing into a primary human T cell a composition comprising achimeric antigen receptor (CAR) to produce a CAR-T cell and (b)contacting the CAR-T cell and a T-cell activator composition comprisingone or more of an anti-human CD3 monospecific tetrameric antibodycomplex, an anti-human CD28 monospecific tetrameric antibody complex, ananti-human CD2 monospecific tetrameric antibody complex and anactivation supplement to produce an activated CAR-T cell, wherein theactivated CAR-T cell expresses one or more cell-surface marker(s) of astem memory T cell (T_(SCM)), thereby producing a CAR-expressing stemmemory T cell (T_(SCM)) (CAR-T_(SCM)). The disclosure provides a methodof producing a plurality of modified stem memory T cells (T_(SCM)),comprising: (a) introducing into a plurality of primary human T cells acomposition comprising a chimeric antigen receptor (CAR) to produce aplurality of CAR-T cells and (b) contacting the plurality of CAR-T cellsand a T-cell activator composition comprising one or more of ananti-human CD3 monospecific tetrameric antibody complex, an anti-humanCD28 monospecific tetrameric antibody complex, an anti-human CD2monospecific tetrameric antibody complex and an activation supplement toproduce a plurality of activated CAR-T cells, wherein at least 2%, 5%,10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 95%, 99% or any percentage in between of the plurality ofactivated CAR-T cells expresses one or more cell-surface marker(s) of astem memory T cell (T_(SCM)), thereby producing a plurality of activatedCAR stem memory T cells (T_(SCM)). In certain embodiments, the methodsfurther comprises the step of: (c) contacting the activated modified Tcell and a T-cell expansion composition comprising one or more of humanserum albumin, recombinant human insulin, human transferrin,2-Mercaptoethanol, Iscove's MDM, and an expansion supplement to producea plurality of expanded modified T-cells, wherein at least 2% of theplurality of expanded modified T-cells expresses one or morecell-surface marker(s) of a stem memory T cell (T_(SCM)). In certainembodiments, the T-cell expansion composition comprises or furthercomprises one or more of octanoic acid, nicotinamide,2,4,7,9-tetramethyl-5-decyn-4,7-diol (TMDD), diisopropyl adipate (DIPA),n-butyl-benzenesulfonamide, 1,2-benzenedicarboxylic acid,bis(2-methylpropyl) ester, palmitic acid, linoleic acid, oleic acid,stearic acid hydrazide, oleamide, a sterol and an alkane. In certainembodiments, the T-cell expansion composition comprises one or more ofoctanoic acid, palmitic acid, linoleic acid, oleic acid and a sterol(e.g. cholesterol). In certain embodiments, the T-cell expansioncomposition comprises one or more of octanoic acid at a concentration ofbetween 0.9 mg/kg to 90 mg/kg, inclusive of the endpoints; palmitic acidat a concentration of between 0.2 mg/kg to 20 mg/kg, inclusive of theendpoints; linoleic acid at a concentration of between 0.2 mg/kg to 20mg/kg, inclusive of the endpoints; oleic acid at a concentration of 0.2mg/kg to 20 mg/kg, inclusive of the endpoints; and a sterol at aconcentration of about 0.1 mg/kg to 10 mg/kg, inclusive of the endpoints(wherein mg/kg=parts per million). In certain embodiments, the T-cellexpansion composition comprises one or more of octanoic acid at aconcentration of about 9 mg/kg, palmitic acid at a concentration ofabout 2 mg/kg, linoleic acid at a concentration of about 2 mg/kg, oleicacid at a concentration of about 2 mg/kg, and a sterol at aconcentration of about 1 mg/kg (wherein mg/kg=parts per million). Incertain embodiments, the T-cell expansion composition comprises one ormore of octanoic acid at a concentration of 9.19 mg/kg, palmitic acid ata concentration of 1.86 mg/kg, linoleic acid at a concentration of about2.12 mg/kg, oleic acid at a concentration of about 2.13 mg/kg, and asterol at a concentration of about 1.01 mg/kg (wherein mg/kg=parts permillion). In certain embodiments, the T-cell expansion compositioncomprises octanoic acid at a concentration of 9.19 mg/kg, palmitic acidat a concentration of 1.86 mg/kg, linoleic acid at a concentration of2.12 mg/kg, oleic acid at a concentration of about 2.13 mg/kg, and asterol at a concentration of 1.01 mg/kg (wherein mg/kg=parts permillion). In certain embodiments, the T-cell expansion compositioncomprises one or more of octanoic acid at a concentration of between 6.4μmol/kg and 640 μmol/kg, inclusive of the endpoints; palmitic acid at aconcentration of between 0.7 μmol/kg and 70 μmol/kg, inclusive of theendpoints; linoleic acid at a concentration of between 0.75 μmol/kg and75 μmol/kg, inclusive of the endpoints; oleic acid at a concentration ofbetween 0.75 μmol/kg and 75 μmol/kg, inclusive of the endpoints; and asterol at a concentration of between 0.25 μmol/kg and 25 μmol/kg,inclusive of the endpoints. In certain embodiments, the T-cell expansioncomposition comprises one or more of octanoic acid at a concentration ofabout 64 μmol/kg, palmitic acid at a concentration of about 7 μmol/kg,linoleic acid at a concentration of about 7.5 μmol/kg, oleic acid at aconcentration of about 7.5 μmol/kg and a sterol at a concentration ofabout 2.5 μmol/kg. In certain embodiments, the T-cell expansioncomposition comprises one or more of octanoic acid at a concentration ofabout 63.75 μmol/kg, palmitic acid at a concentration of about 7.27μmol/kg, linoleic acid at a concentration of about 7.57 μmol/kg, oleicacid at a concentration of about 7.56 μmol/kg and a sterol at aconcentration of about 2.61 μmol/kg. In certain embodiments, the T-cellexpansion composition comprises octanoic acid at a concentration ofabout 63.75 μmol/kg, palmitic acid at a concentration of about 7.27μmol/kg, linoleic acid at a concentration of about 7.57 μmol/kg, oleicacid at a concentration of 7.56 μmol/kg and a sterol at a concentrationof 2.61 μmol/kg. In certain embodiments, at least 2%, 5%, 10%, 15%, 20%,25%, 30%, 35%, 40%, 45%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%,99% or any percentage in between of the plurality of expanded modifiedT-cells expresses cell-surface marker(s) of a stem memory T cell(T_(SCM)). In certain embodiments, at least 60% of the plurality ofexpanded modified T-cells expresses cell-surface marker(s) of a stemmemory T cell (T_(SCM)). In certain embodiments, the method furthercomprises the step of: (d) enriching the plurality of expanded modifiedT-cells to produce a composition comprising at least 2%, 5%, 10%, 15%,20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,95%, 99% or any percentage in between of modified T-cells that expresscell-surface marker(s) of a stem memory T cell (T_(SCM)). In certainembodiments, the method further comprises the step of: (d) enriching theplurality of expanded modified T-cells to produce a compositioncomprising at least 60% of modified T-cells that express cell-surfacemarker(s) of a stem memory T cell (T_(SCM)). In certain embodiments, theenriching step further comprises isolating modified T-cells that expressone or more cell-surface marker(s) of a stem memory T cell (T_(SCM))from the plurality of enriched modified T-cells. In certain embodiments,the enriching step further comprises contacting the isolated modifiedT_(SCM) and a T-cell expansion composition comprising one or more ofhuman serum albumin, recombinant human insulin, human transferrin,2-Mercaptoethanol, Iscove's MDM, and an expansion supplement to producea plurality of expanded enriched modified T_(SCM). In certainembodiments, the T-cell expansion composition further comprises one ormore of octanoic acid, nicotinamide,2,4,7,9-tetramethyl-5-decyn-4,7-diol (TMDD), diisopropyl adipate (DIPA),n-butyl-benzenesulfonamide, 1,2-benzenedicarboxylic acid,bis(2-methylpropyl) ester, palmitic acid, linoleic acid, oleic acid,stearic acid hydrazide, oleamide, a sterol and an alkane. In certainembodiments, the T-cell expansion composition comprises one or more ofoctanoic acid, palmitic acid, linoleic acid, oleic acid and a sterol(e.g. cholesterol). In certain embodiments, the T-cell expansioncomposition comprises one or more of octanoic acid at a concentration ofbetween 0.9 mg/kg to 90 mg/kg, inclusive of the endpoints; palmitic acidat a concentration of between 0.2 mg/kg to 20 mg/kg, inclusive of theendpoints; linoleic acid at a concentration of between 0.2 mg/kg to 20mg/kg, inclusive of the endpoints; oleic acid at a concentration of 0.2mg/kg to 20 mg/kg, inclusive of the endpoints; and a sterol at aconcentration of about 0.1 mg/kg to 10 mg/kg, inclusive of the endpoints(wherein mg/kg=parts per million). In certain embodiments, the T-cellexpansion composition comprises one or more of octanoic acid at aconcentration of about 9 mg/kg, palmitic acid at a concentration ofabout 2 mg/kg, linoleic acid at a concentration of about 2 mg/kg, oleicacid at a concentration of about 2 mg/kg, and a sterol at aconcentration of about 1 mg/kg (wherein mg/kg=parts per million). Incertain embodiments, the T-cell expansion composition comprises one ormore of octanoic acid at a concentration of 9.19 mg/kg, palmitic acid ata concentration of 1.86 mg/kg, linoleic acid at a concentration of about2.12 mg/kg, oleic acid at a concentration of about 2.13 mg/kg, and asterol at a concentration of about 1.01 mg/kg (wherein mg/kg=parts permillion). In certain embodiments, the T-cell expansion compositioncomprises octanoic acid at a concentration of 9.19 mg/kg, palmitic acidat a concentration of 1.86 mg/kg, linoleic acid at a concentration of2.12 mg/kg, oleic acid at a concentration of about 2.13 mg/kg, and asterol at a concentration of 1.01 mg/kg (wherein mg/kg=parts permillion). In certain embodiments, the T-cell expansion compositioncomprises one or more of octanoic acid at a concentration of between 6.4μmol/kg and 640 μmol/kg, inclusive of the endpoints; palmitic acid at aconcentration of between 0.7 μmol/kg and 70 μmol/kg, inclusive of theendpoints; linoleic acid at a concentration of between 0.75 μmol/kg and75 μmol/kg, inclusive of the endpoints; oleic acid at a concentration ofbetween 0.75 μmol/kg and 75 μmol/kg, inclusive of the endpoints; and asterol at a concentration of between 0.25 μmol/kg and 25 μmol/kg,inclusive of the endpoints. In certain embodiments, the T-cell expansioncomposition comprises one or more of octanoic acid at a concentration ofabout 64 μmol/kg, palmitic acid at a concentration of about 7 μmol/kg,linoleic acid at a concentration of about 7.5 μmol/kg, oleic acid at aconcentration of about 7.5 μmol/kg and a sterol at a concentration ofabout 2.5 μmol/kg. In certain embodiments, the T-cell expansioncomposition comprises one or more of octanoic acid at a concentration ofabout 63.75 μmol/kg, palmitic acid at a concentration of about 7.27μmol/kg, linoleic acid at a concentration of about 7.57 μmol/kg, oleicacid at a concentration of about 7.56 μmol/kg and a sterol at aconcentration of about 2.61 μmol/kg. In certain embodiments, the T-cellexpansion composition comprises octanoic acid at a concentration ofabout 63.75 μmol/kg, palmitic acid at a concentration of about 7.27μmol/kg, linoleic acid at a concentration of about 7.57 μmol/kg, oleicacid at a concentration of 7.56 μmol/kg and a sterol at a concentrationof 2.61 μmol/kg.

The disclosure provides a method of producing a modified central memoryT cell (T_(CM)), comprising: (a) introducing into a primary human T cella composition comprising an antigen receptor to produce a modified Tcell, wherein a transposon comprises the antigen receptor, and (b)contacting the modified T cell and a T-cell activator compositioncomprising one or more of an anti-human CD3 monospecific tetramericantibody complex, an anti-human CD28 monospecific tetrameric antibodycomplex and an activation supplement to produce an activated modifiedT-cell, wherein the activated modified-T cell expresses one or morecell-surface marker(s) of a central memory T cell (T_(CM)), therebyproducing a modified central memory T cell (T_(CM)). The disclosureprovides a method of producing a plurality of modified central memory Tcells (T_(CM)), comprising: (a) introducing into a plurality of primaryhuman T cells a composition comprising an antigen receptor to produce aplurality of modified T cells, wherein a transposon comprises theantigen receptor, and (b) contacting the plurality of modified T cellsand a T-cell activator composition comprising one or more of ananti-human CD3 monospecific tetrameric antibody complex, an anti-humanCD28 monospecific tetrameric antibody complex and an activationsupplement to produce a plurality of activated modified T-cells, whereinat least 25%, 50%, 60%, 75%, 80%, 85%, 90%, 95% or 99% of the pluralityof activated modified −T cells expresses one or more cell-surfacemarker(s) of a central memory T cell (T_(CM)), thereby producing amodified central memory T cell (T_(CM)). In certain embodiments of thismethod, at least 60% of the plurality of activated modified −T cellsexpresses one or more cell-surface marker(s) of a central memory T cell(T_(CM)). In certain embodiments of this method, the T-cell activatorcomposition of (b) further comprises an anti-human CD2 monospecifictetrameric antibody complex. In certain embodiments, the methods furthercomprises the step of: (c) contacting the activated modified T cell anda T-cell expansion composition comprising one or more of human serumalbumin, recombinant human insulin, human transferrin,2-Mercaptoethanol, Iscove's MDM, and an expansion supplement to producea plurality of expanded modified T-cells, wherein at least 2% of theplurality of expanded modified T-cells expresses one or morecell-surface marker(s) of a central memory T cell (T_(CM)). In certainembodiments, the T-cell expansion composition comprises or furthercomprises one or more of octanoic acid, nicotinamide,2,4,7,9-tetramethyl-5-decyn-4,7-diol (TMDD), diisopropyl adipate (DIPA),n-butyl-benzenesulfonamide, 1,2-benzenedicarboxylic acid,bis(2-methylpropyl) ester, palmitic acid, linoleic acid, oleic acid,stearic acid hydrazide, oleamide, a sterol and an alkane. In certainembodiments, the T-cell expansion composition comprises one or more ofoctanoic acid, palmitic acid, linoleic acid, oleic acid and a sterol(e.g. cholesterol). In certain embodiments, the T-cell expansioncomposition comprises one or more of octanoic acid at a concentration ofbetween 0.9 mg/kg to 90 mg/kg, inclusive of the endpoints; palmitic acidat a concentration of between 0.2 mg/kg to 20 mg/kg, inclusive of theendpoints; linoleic acid at a concentration of between 0.2 mg/kg to 20mg/kg, inclusive of the endpoints; oleic acid at a concentration of 0.2mg/kg to 20 mg/kg, inclusive of the endpoints; and a sterol at aconcentration of about 0.1 mg/kg to 10 mg/kg, inclusive of the endpoints(wherein mg/kg=parts per million). In certain embodiments, the T-cellexpansion composition comprises one or more of octanoic acid at aconcentration of about 9 mg/kg, palmitic acid at a concentration ofabout 2 mg/kg, linoleic acid at a concentration of about 2 mg/kg, oleicacid at a concentration of about 2 mg/kg, and a sterol at aconcentration of about 1 mg/kg (wherein mg/kg=parts per million). Incertain embodiments, the T-cell expansion composition comprises one ormore of octanoic acid at a concentration of 9.19 mg/kg, palmitic acid ata concentration of 1.86 mg/kg, linoleic acid at a concentration of about2.12 mg/kg, oleic acid at a concentration of about 2.13 mg/kg, and asterol at a concentration of about 1.01 mg/kg (wherein mg/kg=parts permillion). In certain embodiments, the T-cell expansion compositioncomprises octanoic acid at a concentration of 9.19 mg/kg, palmitic acidat a concentration of 1.86 mg/kg, linoleic acid at a concentration of2.12 mg/kg, oleic acid at a concentration of about 2.13 mg/kg, and asterol at a concentration of 1.01 mg/kg (wherein mg/kg=parts permillion). In certain embodiments, the T-cell expansion compositioncomprises one or more of octanoic acid at a concentration of between 6.4μmol/kg and 640 μmol/kg, inclusive of the endpoints; palmitic acid at aconcentration of between 0.7 μmol/kg and 70 μmol/kg, inclusive of theendpoints; linoleic acid at a concentration of between 0.75 μmol/kg and75 μmol/kg, inclusive of the endpoints; oleic acid at a concentration ofbetween 0.75 μmol/kg and 75 μmol/kg, inclusive of the endpoints; and asterol at a concentration of between 0.25 μmol/kg and 25 μmol/kg,inclusive of the endpoints. In certain embodiments, the T-cell expansioncomposition comprises one or more of octanoic acid at a concentration ofabout 64 μmol/kg, palmitic acid at a concentration of about 7 μmol/kg,linoleic acid at a concentration of about 7.5 μmol/kg, oleic acid at aconcentration of about 7.5 μmol/kg and a sterol at a concentration ofabout 2.5 μmol/kg. In certain embodiments, the T-cell expansioncomposition comprises one or more of octanoic acid at a concentration ofabout 63.75 μmol/kg, palmitic acid at a concentration of about 7.27μmol/kg, linoleic acid at a concentration of about 7.57 μmol/kg, oleicacid at a concentration of about 7.56 μmol/kg and a sterol at aconcentration of about 2.61 μmol/kg. In certain embodiments, the T-cellexpansion composition comprises octanoic acid at a concentration ofabout 63.75 μmol/kg, palmitic acid at a concentration of about 7.27μmol/kg, linoleic acid at a concentration of about 7.57 μmol/kg, oleicacid at a concentration of 7.56 μmol/kg and a sterol at a concentrationof 2.61 μmol/kg. In certain embodiments, at least 2%, 5%, 10%, 15%, 20%,25%, 30%, 35%, 40%, 45%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%,99% or any percentage in between of the plurality of expanded modifiedT-cells expresses cell-surface marker(s) of a central memory T cell(T_(CM)). In certain embodiments, at least 60% of the plurality ofexpanded modified T-cells expresses cell-surface marker(s) of a centralmemory T cell (T_(CM)). In certain embodiments, the method furthercomprises the step of: (d) enriching the plurality of expanded modifiedT-cells to produce a composition comprising at least 2%, 5%, 10%, 15%,20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,95%, 99% or any percentage in between of modified T-cells that expresscell-surface marker(s) of a central memory T cell (T_(CM)). In certainembodiments, the method further comprises the step of: (d) enriching theplurality of expanded modified T-cells to produce a compositioncomprising at least 60% of modified T-cells that express cell-surfacemarker(s) of a central memory T cell (T_(CM)). In certain embodiments,the enriching step further comprises isolating modified T-cells thatexpress one or more cell-surface marker(s) of a central memory T cell(T_(CM)) from the plurality of enriched modified T-cells. In certainembodiments, the enriching step further comprises contacting theisolated modified T_(CM) and a T-cell expansion composition comprisingone or more of human serum albumin, recombinant human insulin, humantransferrin, 2-Mercaptoethanol, Iscove's MDM, and an expansionsupplement to produce a plurality of expanded enriched modified T_(CM).In certain embodiments, the T-cell expansion composition furthercomprises one or more of octanoic acid, nicotinamide,2,4,7,9-tetramethyl-5-decyn-4,7-diol (TMDD), diisopropyl adipate (DIPA),n-butyl-benzenesulfonamide, 1,2-benzenedicarboxylic acid,bis(2-methylpropyl) ester, palmitic acid, linoleic acid, oleic acid,stearic acid hydrazide, oleamide, a sterol and an alkane. In certainembodiments, the T-cell expansion composition comprises one or more ofoctanoic acid, palmitic acid, linoleic acid, oleic acid and a sterol(e.g. cholesterol). In certain embodiments, the T-cell expansioncomposition comprises one or more of octanoic acid at a concentration ofbetween 0.9 mg/kg to 90 mg/kg, inclusive of the endpoints; palmitic acidat a concentration of between 0.2 mg/kg to 20 mg/kg, inclusive of theendpoints; linoleic acid at a concentration of between 0.2 mg/kg to 20mg/kg, inclusive of the endpoints; oleic acid at a concentration of 0.2mg/kg to 20 mg/kg, inclusive of the endpoints; and a sterol at aconcentration of about 0.1 mg/kg to 10 mg/kg, inclusive of the endpoints(wherein mg/kg=parts per million). In certain embodiments, the T-cellexpansion composition comprises one or more of octanoic acid at aconcentration of about 9 mg/kg, palmitic acid at a concentration ofabout 2 mg/kg, linoleic acid at a concentration of about 2 mg/kg, oleicacid at a concentration of about 2 mg/kg, and a sterol at aconcentration of about 1 mg/kg (wherein mg/kg=parts per million). Incertain embodiments, the T-cell expansion composition comprises one ormore of octanoic acid at a concentration of 9.19 mg/kg, palmitic acid ata concentration of 1.86 mg/kg, linoleic acid at a concentration of about2.12 mg/kg, oleic acid at a concentration of about 2.13 mg/kg, and asterol at a concentration of about 1.01 mg/kg (wherein mg/kg=parts permillion). In certain embodiments, the T-cell expansion compositioncomprises octanoic acid at a concentration of 9.19 mg/kg, palmitic acidat a concentration of 1.86 mg/kg, linoleic acid at a concentration of2.12 mg/kg, oleic acid at a concentration of about 2.13 mg/kg, and asterol at a concentration of 1.01 mg/kg (wherein mg/kg=parts permillion). In certain embodiments, the T-cell expansion compositioncomprises one or more of octanoic acid at a concentration of between 6.4μmol/kg and 640 μmol/kg, inclusive of the endpoints; palmitic acid at aconcentration of between 0.7 μmol/kg and 70 μmol/kg, inclusive of theendpoints; linoleic acid at a concentration of between 0.75 μmol/kg and75 μmol/kg, inclusive of the endpoints; oleic acid at a concentration ofbetween 0.75 μmol/kg and 75 μmol/kg, inclusive of the endpoints; and asterol at a concentration of between 0.25 μmol/kg and 25 μmol/kg,inclusive of the endpoints. In certain embodiments, the T-cell expansioncomposition comprises one or more of octanoic acid at a concentration ofabout 64 μmol/kg, palmitic acid at a concentration of about 7 μmol/kg,linoleic acid at a concentration of about 7.5 μmol/kg, oleic acid at aconcentration of about 7.5 μmol/kg and a sterol at a concentration ofabout 2.5 μmol/kg. In certain embodiments, the T-cell expansioncomposition comprises one or more of octanoic acid at a concentration ofabout 63.75 μmol/kg, palmitic acid at a concentration of about 7.27μmol/kg, linoleic acid at a concentration of about 7.57 μmol/kg, oleicacid at a concentration of about 7.56 μmol/kg and a sterol at aconcentration of about 2.61 μmol/kg. In certain embodiments, the T-cellexpansion composition comprises octanoic acid at a concentration ofabout 63.75 μmol/kg, palmitic acid at a concentration of about 7.27μmol/kg, linoleic acid at a concentration of about 7.57 μmol/kg, oleicacid at a concentration of 7.56 μmol/kg and a sterol at a concentrationof 2.61 μmol/kg.

The disclosure provides a method of producing a composition comprising aplurality of modified stem memory T-cells (T_(SCM)) and a plurality ofmodified central memory T-cells (T_(CM)), comprising: (a) introducinginto a plurality of primary human T cells a composition comprising anantigen receptor to produce a composition comprising a plurality ofmodified stem memory T-cells (T_(SCM)) and a plurality of modifiedcentral memory T-cells (T_(CM)), wherein a transposon comprises theantigen receptor, and (b) contacting the composition and a T-cellactivator composition comprising one or more of an anti-human CD3monospecific tetrameric antibody complex, an anti-human CD28monospecific tetrameric antibody complex and an activation supplement toproduce a composition comprising a plurality of activated modified stemmemory T-cells (T_(SCM)) and a plurality of activated modified centralmemory T-cells (T_(CM)), wherein the plurality of activated modifiedT_(SCM) expresses one or more CD62L, CD45RA, CD28, CCR7, CD127, CD45RO,CD95, CD95 and IL-2Rβ and the plurality of activated modified T_(CM)expresses one or more CD45RO, CD95, IL-2Rβ, CCR7, and CD62L, therebyproducing a composition comprising a plurality of modified T_(SCM) and aplurality of modified T_(CM). In certain embodiments of this method, theT-cell activator composition of (b) further comprises an anti-human CD2monospecific tetrameric antibody complex. In certain embodiments, themethods further comprises the step of: (c) contacting the compositionand a T-cell expansion composition comprising one or more of human serumalbumin, recombinant human insulin, human transferrin,2-Mercaptoethanol, Iscove's MDM, and an expansion supplement to producea plurality of expanded modified T-cells, wherein at least 2% of thecomposition comprising a plurality of expanded modified T-cellsexpresses one or more cell-surface marker(s) of a stem memory T cell(T_(SCM)). In certain embodiments, the methods further comprises thestep of: (c) contacting the composition and a T-cell expansioncomposition comprising one or more of human serum albumin, recombinanthuman insulin, human transferrin, 2-Mercaptoethanol, Iscove's MDM, andan expansion supplement to produce a plurality of expanded modifiedT-cells, wherein at least 2% of the composition comprising a pluralityof expanded modified T-cells expresses one or more cell-surfacemarker(s) of a central memory T cell (T_(CM)). In certain embodiments,the T-cell expansion composition comprises or further comprises one ormore of octanoic acid, nicotinamide,2,4,7,9-tetramethyl-5-decyn-4,7-diol (TMDD), diisopropyl adipate (DIPA),n-butyl-benzenesulfonamide, 1,2-benzenedicarboxylic acid,bis(2-methylpropyl) ester, palmitic acid, linoleic acid, oleic acid,stearic acid hydrazide, oleamide, a sterol and an alkane. In certainembodiments, the T-cell expansion composition comprises one or more ofoctanoic acid, palmitic acid, linoleic acid, oleic acid and a sterol(e.g. cholesterol). In certain embodiments, the T-cell expansioncomposition comprises one or more of octanoic acid at a concentration ofbetween 0.9 mg/kg to 90 mg/kg, inclusive of the endpoints; palmitic acidat a concentration of between 0.2 mg/kg to 20 mg/kg, inclusive of theendpoints; linoleic acid at a concentration of between 0.2 mg/kg to 20mg/kg, inclusive of the endpoints; oleic acid at a concentration of 0.2mg/kg to 20 mg/kg, inclusive of the endpoints; and a sterol at aconcentration of about 0.1 mg/kg to 10 mg/kg, inclusive of the endpoints(wherein mg/kg=parts per million). In certain embodiments, the T-cellexpansion composition comprises one or more of octanoic acid at aconcentration of about 9 mg/kg, palmitic acid at a concentration ofabout 2 mg/kg, linoleic acid at a concentration of about 2 mg/kg, oleicacid at a concentration of about 2 mg/kg, and a sterol at aconcentration of about 1 mg/kg (wherein mg/kg=parts per million). Incertain embodiments, the T-cell expansion composition comprises one ormore of octanoic acid at a concentration of 9.19 mg/kg, palmitic acid ata concentration of 1.86 mg/kg, linoleic acid at a concentration of about2.12 mg/kg, oleic acid at a concentration of about 2.13 mg/kg, and asterol at a concentration of about 1.01 mg/kg (wherein mg/kg=parts permillion). In certain embodiments, the T-cell expansion compositioncomprises octanoic acid at a concentration of 9.19 mg/kg, palmitic acidat a concentration of 1.86 mg/kg, linoleic acid at a concentration of2.12 mg/kg, oleic acid at a concentration of about 2.13 mg/kg, and asterol at a concentration of 1.01 mg/kg (wherein mg/kg=parts permillion). In certain embodiments, the T-cell expansion compositioncomprises one or more of octanoic acid at a concentration of between 6.4μmol/kg and 640 μmol/kg, inclusive of the endpoints; palmitic acid at aconcentration of between 0.7 μmol/kg and 70 μmol/kg, inclusive of theendpoints; linoleic acid at a concentration of between 0.75 μmol/kg and75 μmol/kg, inclusive of the endpoints; oleic acid at a concentration ofbetween 0.75 μmol/kg and 75 μmol/kg, inclusive of the endpoints; and asterol at a concentration of between 0.25 μmol/kg and 25 μmol/kg,inclusive of the endpoints. In certain embodiments, the T-cell expansioncomposition comprises one or more of octanoic acid at a concentration ofabout 64 μmol/kg, palmitic acid at a concentration of about 7 μmol/kg,linoleic acid at a concentration of about 7.5 μmol/kg, oleic acid at aconcentration of about 7.5 μmol/kg and a sterol at a concentration ofabout 2.5 μmol/kg. In certain embodiments, the T-cell expansioncomposition comprises one or more of octanoic acid at a concentration ofabout 63.75 μmol/kg, palmitic acid at a concentration of about 7.27μmol/kg, linoleic acid at a concentration of about 7.57 μmol/kg, oleicacid at a concentration of about 7.56 μmol/kg and a sterol at aconcentration of about 2.61 μmol/kg. In certain embodiments, the T-cellexpansion composition comprises octanoic acid at a concentration ofabout 63.75 μmol/kg, palmitic acid at a concentration of about 7.27μmol/kg, linoleic acid at a concentration of about 7.57 μmol/kg, oleicacid at a concentration of 7.56 μmol/kg and a sterol at a concentrationof 2.61 μmol/kg. In certain embodiments, at least 2%, 5%, 10%, 15%, 20%,25%, 30%, 35%, 40%, 45%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%,99% or any percentage in between of cells the composition comprising aplurality of expanded modified T_(SCM) and a plurality of expandedmodified T_(CM) expresses cell-surface marker(s) of a stem memory T cell(T_(SCM)). In certain embodiments, at least 2%, 5%, 10%, 15%, 20%, 25%,30%, 35%, 40%, 45%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% orany percentage in between of cells the composition comprising aplurality of expanded modified T_(SCM) and a plurality of expandedmodified T_(CM) expresses cell-surface marker(s) of a central memory Tcell (T_(CM)). In certain embodiments, the method further comprises thestep of: (d) enriching the composition to produce a compositioncomprising at least 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%,60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or any percentage in betweenof modified T-cells that express cell-surface marker(s) of a stem memoryT cell (T_(SCM)). In certain embodiments, the method further comprisesthe step of: (d) enriching the composition to produce a compositioncomprising at least 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%,60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or any percentage in betweenof modified T-cells that express cell-surface marker(s) of a centralmemory T cell (T_(CM)). In certain embodiments, the enriching stepfurther comprises isolating modified T-cells that express one or morecell-surface marker(s) of a stem memory T cell (T_(SCM)) from thecomposition or isolating modified T-cells that express one or morecell-surface marker(s) of a central memory T cell (T_(CM)) from thecomposition. In certain embodiments, the enriching step furthercomprises isolating modified T-cells that express one or morecell-surface marker(s) of a stem memory T cell (T_(SCM)) from thecomposition and isolating modified T-cells that express one or morecell-surface marker(s) of a central memory T cell (T_(CM)) from thecomposition. In certain embodiments, the enriching step furthercomprises contacting the isolated modified T_(SCM) and/or T_(CM) and aT-cell expansion composition comprising one or more of human serumalbumin, recombinant human insulin, human transferrin,2-Mercaptoethanol, Iscove's MDM, and an expansion supplement to producea composition comprising a plurality of expanded enriched modifiedT_(SCM) and/or T_(CM). In certain embodiments, the T-cell expansioncomposition further comprises one or more of octanoic acid,nicotinamide, 2,4,7,9-tetramethyl-5-decyn-4,7-diol (TMDD), diisopropyladipate (DIPA), n-butyl-benzenesulfonamide, 1,2-benzenedicarboxylicacid, bis(2-methylpropyl) ester, palmitic acid, linoleic acid, oleicacid, stearic acid hydrazide, oleamide, a sterol and an alkane. Incertain embodiments, the T-cell expansion composition comprises one ormore of octanoic acid, palmitic acid, linoleic acid, oleic acid and asterol (e.g. cholesterol). In certain embodiments, the T-cell expansioncomposition comprises one or more of octanoic acid at a concentration ofbetween 0.9 mg/kg to 90 mg/kg, inclusive of the endpoints; palmitic acidat a concentration of between 0.2 mg/kg to 20 mg/kg, inclusive of theendpoints; linoleic acid at a concentration of between 0.2 mg/kg to 20mg/kg, inclusive of the endpoints; oleic acid at a concentration of 0.2mg/kg to 20 mg/kg, inclusive of the endpoints; and a sterol at aconcentration of about 0.1 mg/kg to 10 mg/kg, inclusive of the endpoints(wherein mg/kg=parts per million). In certain embodiments, the T-cellexpansion composition comprises one or more of octanoic acid at aconcentration of about 9 mg/kg, palmitic acid at a concentration ofabout 2 mg/kg, linoleic acid at a concentration of about 2 mg/kg, oleicacid at a concentration of about 2 mg/kg, and a sterol at aconcentration of about 1 mg/kg (wherein mg/kg=parts per million). Incertain embodiments, the T-cell expansion composition comprises one ormore of octanoic acid at a concentration of 9.19 mg/kg, palmitic acid ata concentration of 1.86 mg/kg, linoleic acid at a concentration of about2.12 mg/kg, oleic acid at a concentration of about 2.13 mg/kg, and asterol at a concentration of about 1.01 mg/kg (wherein mg/kg=parts permillion). In certain embodiments, the T-cell expansion compositioncomprises octanoic acid at a concentration of 9.19 mg/kg, palmitic acidat a concentration of 1.86 mg/kg, linoleic acid at a concentration of2.12 mg/kg, oleic acid at a concentration of about 2.13 mg/kg, and asterol at a concentration of 1.01 mg/kg (wherein mg/kg=parts permillion). In certain embodiments, the T-cell expansion compositioncomprises one or more of octanoic acid at a concentration of between 6.4μmol/kg and 640 μmol/kg, inclusive of the endpoints; palmitic acid at aconcentration of between 0.7 μmol/kg and 70 μmol/kg, inclusive of theendpoints; linoleic acid at a concentration of between 0.75 μmol/kg and75 μmol/kg, inclusive of the endpoints; oleic acid at a concentration ofbetween 0.75 μmol/kg and 75 μmol/kg, inclusive of the endpoints; and asterol at a concentration of between 0.25 μmol/kg and 25 μmol/kg,inclusive of the endpoints. In certain embodiments, the T-cell expansioncomposition comprises one or more of octanoic acid at a concentration ofabout 64 μmol/kg, palmitic acid at a concentration of about 7 μmol/kg,linoleic acid at a concentration of about 7.5 μmol/kg, oleic acid at aconcentration of about 7.5 μmol/kg and a sterol at a concentration ofabout 2.5 μmol/kg. In certain embodiments, the T-cell expansioncomposition comprises one or more of octanoic acid at a concentration ofabout 63.75 μmol/kg, palmitic acid at a concentration of about 7.27μmol/kg, linoleic acid at a concentration of about 7.57 μmol/kg, oleicacid at a concentration of about 7.56 μmol/kg and a sterol at aconcentration of about 2.61 μmol/kg. In certain embodiments, the T-cellexpansion composition comprises octanoic acid at a concentration ofabout 63.75 μmol/kg, palmitic acid at a concentration of about 7.27μmol/kg, linoleic acid at a concentration of about 7.57 μmol/kg, oleicacid at a concentration of 7.56 μmol/kg and a sterol at a concentrationof 2.61 μmol/kg. In certain embodiments of this method, the modifiedstem memory T-cells (T_(SCM)) comprise at least 1%, 2%, 5%, 7%, 10%,15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 95%, 97%, 99% or any percentage of cells in between of thetotal number of cells of the composition. In certain embodiments of thismethod, the modified central memory T-cells (T_(CM)) comprise at least1%, 2%, 5%, 7%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%,65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99% or any percentage of cellsin between of the total number of cells of the composition. In certainembodiments of this method, the modified stem memory T-cells (T_(SCM))comprise at least 10% of the total number of cells of the compositionand the modified central memory T-cells (T_(CM)) comprise at least 90%of the total number of cells of the composition. In certain embodimentsof this method, the modified stem memory T-cells (T_(SCM)) comprise atleast 90% of the total number of cells of the composition and themodified central memory T-cells (T_(CM)) comprise at least 10% of thetotal number of cells of the composition. In certain embodiments of thismethod, the modified stem memory T-cells (T_(SCM)) comprise at least 20%of the total number of cells of the composition and the modified centralmemory T-cells (T_(CM)) comprise at least 80% of the total number ofcells of the composition. In certain embodiments of this method, themodified stem memory T-cells (T_(SCM)) comprise at least 80% of thetotal number of cells of the composition and the modified central memoryT-cells (T_(CM)) comprise at least 20% of the total number of cells ofthe composition. In certain embodiments of this method, the modifiedstem memory T-cells (T_(SCM)) comprise at least 30% of the total numberof cells of the composition and the modified central memory T-cells(T_(CM)) comprise at least 70% of the total number of cells of thecomposition. In certain embodiments of this method, the modified stemmemory T-cells (T_(SCM)) comprise at least 70% of the total number ofcells of the composition and the modified central memory T-cells(T_(CM)) comprise at least 30% of the total number of cells of thecomposition. In certain embodiments of this method, the modified stemmemory T-cells (T_(SCM)) comprise at least 40% of the total number ofcells of the composition and the modified central memory T-cells(T_(CM)) comprise at least 60% of the total number of cells of thecomposition. In certain embodiments of this method, the modified stemmemory T-cells (T_(SCM)) comprise at least 60% of the total number ofcells of the composition and the modified central memory T-cells(T_(CM)) comprise at least 40% of the total number of cells of thecomposition. In certain embodiments of this method, the modified stemmemory T-cells (T_(SCM)) comprise at least 50% of the total number ofcells of the composition and the modified central memory T-cells(T_(CM)) comprise at least 50% of the total number of cells of thecomposition.

In certain embodiments of the methods of the disclosure, including thosewherein the method comprises introducing into a primary human T cell (a)introducing into a primary human T cell a composition comprising anantigen receptor to produce a modified T cell, wherein a transposoncomprises the antigen receptor, and (b) contacting the modified T celland a T-cell activator composition comprising one or more of ananti-human CD3 monospecific tetrameric antibody complex, an anti-humanCD28 monospecific tetrameric antibody complex and an activationsupplement to produce an activated modified T-cell, the method furthercomprises introducing into the primary human T cell (c) a secondtransposon composition comprising a transposon comprising a therapeuticprotein, to produce a modified T cell, wherein the modified T cell iscapable of expressing the therapeutic protein. In certain embodiments,the therapeutic protein is a secretable protein and the method producesa modified T cell capable of secreting the therapeutic protein. Incertain embodiments, the method further comprises introducing atransposase composition. In certain embodiments, the transposasecomposition transposes the transposon of (a) and the second transposon.In certain embodiments, the method comprises introducing a firsttransposase composition and a second transposase composition. In certainembodiments, including those wherein the method comprises introducing afirst transposase composition and a second transposase composition, thefirst transposase composition transposes the transposon of (a) and thesecond transposase composition transposes the second transposon. Incertain embodiments of this method, the transposon is a plasmid DNAtransposon with a sequence encoding the antigen receptor or thetherapeutic protein flanked by two cis-regulatory insulator elements. Incertain embodiments, the transposon is a piggyBac transposon. In certainembodiments, and, in particular, those embodiments wherein thetransposon is a piggyBac transposon, the transposase is a piggyBac™ or aSuper piggyBac™ (SPB) transposase. In certain embodiments of thismethod, the transposon is a Sleeping Beauty transposon. In certainembodiments, and, in particular, those embodiments wherein thetransposon is a Sleeping Beauty transposon, the transposase is aSleeping Beauty transposase or a hyperactive Sleeping Beauty transposase(SB100X). In certain embodiments of this method, the transposon is aHelraiser transposon. In certain embodiments, and, in particular, thoseembodiments wherein the transposon is a Helraiser transposon, thetransposase is a Helitron transposase. In certain embodiments of thismethod, the transposon is a Tol2 transposon. In certain embodiments,including those embodiments wherein the transposon is a Tol2 transposon,the transposase is a Tol2 transposase.

In certain embodiments of the methods of the disclosure, including thosewherein the method comprises introducing into a primary human T cell (a)introducing into a primary human T cell a composition comprising anantigen receptor to produce a modified T cell, wherein a transposoncomprises the antigen receptor, and (b) contacting the modified T celland a T-cell activator composition comprising one or more of ananti-human CD3 monospecific tetrameric antibody complex, an anti-humanCD28 monospecific tetrameric antibody complex and an activationsupplement to produce an activated modified T-cell, the method furthercomprises introducing into the primary human T cell a sequence encodinga therapeutic protein, to produce a modified T cell, wherein themodified T cell is capable of expressing the therapeutic protein. Incertain embodiments of introducing a sequence encoding a therapeuticprotein, the introducing step comprises a homologous recombination. Incertain embodiments of introducing a sequence encoding a therapeuticprotein, a vector comprises the sequence encoding the therapeuticprotein. In certain embodiments, the vector is a viral vector. Incertain embodiments, the vector is a nanoparticle.

In certain embodiments of the methods of the disclosure, the introducingstep further comprises a composition comprising a genomic editingconstruct. In certain embodiments, the genomic editing constructcomprises a guide RNA and a clustered regularly interspaced shortpalindromic repeats (CRISPR) associated protein 9 (Cas9) DNAendonuclease. In certain embodiments, the genomic editing constructcomprises a DNA binding domain and a type IIS endonuclease. In certainembodiments, the genomic editing construct encodes a fusion protein. Incertain embodiments, the genomic editing construct encodes the DNAbinding domain and the type IIS endonuclease and wherein the expressedDNA binding domain and the expressed type IIS endonuclease arenon-covalently linked. In certain embodiments, including thoseembodiments wherein the genomic editing construct comprises a DNAbinding domain and a type IIS endonuclease, the genomic editingconstruct comprises a sequence derived from a Cas9 endonuclease. Incertain embodiments, including those embodiments wherein the genomicediting construct comprises a DNA binding domain and a type IISendonuclease, the sequence derived from a Cas9 endonuclease is the DNAbinding domain. In certain embodiments, including those embodimentswherein the sequence derived from a Cas9 endonuclease is the DNA bindingdomain, the sequence derived from a Cas9 endonuclease encodes aninactive Cas9. In certain embodiments, including those embodimentswherein the sequence derived from a Cas9 endonuclease is the DNA bindingdomain, the sequence derived from a Cas9 endonuclease encodes atruncated Cas9. In certain embodiments, the sequence derived from a Cas9endonuclease comprises an amino acid substitution of an Alanine (A) foran Aspartic Acid (D) at position 10 (D10A). In certain embodiments, thesequence derived from a Cas9 endonuclease comprises an amino acidsubstitution of an Alanine (A) for a Histidine (H) at position 840(H840A). In certain embodiments, the sequence derived from a Cas9endonuclease comprises dCas9 (SEQ ID NO: 33). In certain embodiments,the sequence derived from a Cas9 endonuclease comprises an amino acidsubstitution of an Alanine (A) for an Asparagine (N) at position 580(N580A). In certain embodiments, the sequence derived from a Cas9endonuclease comprises dSaCas9 (SEQ ID NO: 32). In certain embodiments,including those embodiments wherein the genomic editing constructcomprises a DNA binding domain and a type IIS endonuclease, the genomicediting construct comprises a sequence derived from a transcriptionactivator-like effector nuclease (TALEN). In certain embodiments,including those embodiments wherein the genomic editing constructcomprises a DNA binding domain and a type IIS endonuclease, the sequencederived from a TALEN is the DNA binding domain. In certain embodiments,the genomic editing construct comprises a TALEN. In certain embodiments,including those embodiments wherein the genomic editing constructcomprises a DNA binding domain and a type IIS endonuclease, the genomicediting construct comprises a sequence derived from a zinc-fingernuclease (ZFN). In certain embodiments, including those embodimentswherein the genomic editing construct comprises a DNA binding domain anda type IIS endonuclease, the sequence derived from a ZFN is the DNAbinding domain. In certain embodiments, the genomic editing constructcomprises a zinc-finger nuclease (ZFN).

In certain embodiments of the methods of the disclosure, the transposonis a plasmid DNA transposon with a sequence encoding the antigenreceptor or the therapeutic protein flanked by two cis-regulatoryinsulator elements. In certain embodiments of this method, theintroducing step further comprises a composition comprising an mRNAsequence encoding a transposase. In certain embodiments, the transposonis a piggyBac transposon. In certain embodiments, and, in particular,those embodiments wherein the transposon is a piggyBac transposon, thetransposase is a Super piggyBac™ (SPB) transposase. In certainembodiments, and, in particular, those embodiments wherein thetransposase is a Super piggyBac™ (SPB) transposase, the sequenceencoding the transposase is an mRNA sequence. In certain embodiments,the piggyBac transposase comprises an amino acid sequence comprising SEQID NO: 4. In certain embodiments, the piggyBac transposase is ahyperactive variant and the hyperactive variant comprises an amino acidsubstitution at one or more of positions 30, 165, 282 and 538 of SEQ IDNO: 4. In certain embodiments, the amino acid substitution at position30 of SEQ ID NO: 4 is a substitution of a valine (V) for an isoleucine(I) (I30V). In certain embodiments, the amino acid substitution atposition 165 of SEQ ID NO: 4 is a substitution of a serine (S) for aglycine (G) (G165S). In certain embodiments, the amino acid substitutionat position 282 of SEQ ID NO: 4 is a substitution of a valine (V) for amethionine (M) (M282V). In certain embodiments, the amino acidsubstitution at position 538 of SEQ ID NO: 4 is a substitution of alysine (K) for an asparagine (N) (N538K). In certain embodiments, theSuper piggyBac (SPB) transposase comprises an amino acid sequencecomprising SEQ ID NO: 5. In certain embodiments, the transposon is aSleeping Beauty transposon. In certain embodiments, and, in particular,those embodiments wherein the transposon is a Sleeping Beautytransposon, the transposase is a Sleeping Beauty transposase or ahyperactive Sleeping Beauty transposase (SB100X). In certainembodiments, the transposon is a Helraiser transposon. In certainembodiments, in particular those embodiments wherein the transposon is aHelraiser transposon, the transposase is a Helitron transposase. Incertain embodiments, the transposon is a Tol2 transposon. In certainembodiments, in particular those embodiments wherein the transposon is aTol2 transposon, the transposase is a Tol2 transposase. In certainembodiments, the sequence encoding the transposase is an mRNA sequence.In certain embodiments, the transposon may be derived or recombined fromany species. Alternatively, or in addition, the transposon may besynthetic.

In certain embodiments of the methods of the disclosure, the transposonfurther comprises a selection gene. In certain embodiments, the T-cellexpansion composition further comprises a selection agent.

In certain embodiments of the methods of the disclosure, the antigenreceptor is a T-cell receptor. In certain embodiments, the T-cellreceptor is naturally-occurring. In certain embodiments, the T-cellreceptor is not naturally-occurring. In certain embodiments, and, inparticular, those embodiments wherein the T-cell receptor is notnaturally-occurring, the T-cell receptor comprises one or moremutation(s) compared to a wild-type T-cell receptor. In certainembodiments, and, in particular, those embodiments wherein the T-cellreceptor is not naturally-occurring, the T-cell receptor is arecombinant T-cell receptor. In certain embodiments of this method, theantigen receptor is a Chimeric Antigen Receptor (CAR). In certainembodiments, the CAR is a CARTyrin. In certain embodiments, the CARcomprises one or more VHH sequence(s). In certain embodiments, the CARis a VCAR.

In certain embodiments of the methods of the disclosure, thecell-surface markers of the modified T_(SCM) comprise CD62L and CD45RA.In certain embodiments, the cell-surface markers of the modified T_(SCM)comprise one or more of CD62L, CD45RA, CD28, CCR7, CD127, CD45RO, CD95,CD95 and IL-2Rβ. In certain embodiments, the cell-surface markers of themodified T_(SCM) comprise one or more of CD45RA, CD95, IL-2Rβ, CR7, andCD62L.

In certain embodiments of the methods of the disclosure, the pluralityof expanded modified T-cells comprises a naïve T-cell (modified T_(N))and the cell-surface markers of the CAR-T_(N) comprise one or more ofCD45RA, CCR7 and CD62L. In certain embodiments, the plurality ofexpanded modified T-cells comprises a central memory T-cell (modifiedT_(CM)) and the cell-surface markers of the CAR-T_(CM) comprise one ormore of CD45RO, CD95, CCR7, and CD62L. In certain embodiments, theplurality of expanded modified T-cells comprises an effector memoryT-cell (modified T_(EM)) and the cell-surface markers of the CAR-T_(EM)comprise one or more of CD45RO, CD95, and IL-2Rβ. In certainembodiments, plurality of expanded modified T-cells comprises aneffector T-cell (modified T_(EFF)) and the cell-surface markers of theCAR-T_(EFF) comprise one or more of CD45RA, CD95, and IL-2Rβ.

In certain embodiments of the methods of the disclosure, the pluralityof expanded modified T-cells comprises a central memory T-cell (modifiedT_(CM)) and the cell-surface markers of the CAR-T_(CM) comprise one ormore of CD45RO, CD95, IL-2Rβ, CCR7, and CD62L. In certain embodiments,the most abundant cell in the plurality of expanded modified T-cells isa central memory T-cell (modified T_(CM)) and the cell-surface markersof the CAR-T_(CM) comprise one or more of CD45RO, CD95, IL-2Rβ, CCR7,and CD62L. In certain embodiments, wherein the most abundant cell in theplurality of expanded modified T-cells is a central memory T-cell(modified T_(CM)), the plurality of expanded modified T-cells comprisesa T_(SCM) cell and the cell-surface markers of the T_(SCM) cell compriseone or more of CD62L, CD45RA, CD28, CCR7, CD127, CD45RO, CD95, CD95 andIL-2Rβ.

The disclosure provides a method of producing a modified stem memory Tcell (T_(SCM)), comprising: (a) introducing into a primary human T cella composition comprising a chimeric antigen receptor (CAR) to produce aCAR-T cell and (b) contacting the CAR-T cell and a T-cell activatorcomposition comprising one or more of an anti-human CD3 monospecifictetrameric antibody complex, an anti-human CD28 monospecific tetramericantibody complex, an anti-human CD2 monospecific tetrameric antibodycomplex and an activation supplement to produce an activated CAR-T cell,wherein the activated CAR-T cell expresses one or more cell-surfacemarker(s) of a stem memory T cell (T_(SCM)), thereby producing aCAR-expressing stem memory T cell (T_(SCM)) (CAR-T_(SCM)). Thedisclosure provides a method of producing a plurality of modified stemmemory T cells (T_(SCM)), comprising: (a) introducing into a pluralityof primary human T cells a composition comprising a chimeric antigenreceptor (CAR) to produce a plurality of CAR-T cells and (b) contactingthe plurality of CAR-T cells and a T-cell activator compositioncomprising one or more of an anti-human CD3 monospecific tetramericantibody complex, an anti-human CD28 monospecific tetrameric antibodycomplex, an anti-human CD2 monospecific tetrameric antibody complex andan activation supplement to produce a plurality of activated CAR-Tcells, wherein at least 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%,50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or any percentage inbetween of the plurality of activated CAR-T cells expresses one or morecell-surface marker(s) of a stem memory T cell (T_(SCM)), therebyproducing a plurality of activated CAR stem memory T cells (T_(SCM)). Incertain embodiments, the method produces a plurality of activated CAR-Tcells, wherein at least 25% of the plurality of activated CAR-T cellsexpresses one or more cell-surface marker(s) of a stem memory T cell(T_(SCM)), thereby producing a plurality of activated CAR stem memory Tcells (T_(SCM)). In certain embodiments, the method produces a pluralityof activated CAR-T cells, wherein at least 50% of the plurality ofactivated CAR-T cells expresses one or more cell-surface marker(s) of astem memory T cell (T_(SCM)), thereby producing a plurality of activatedCAR stem memory T cells (T_(SCM)). In certain embodiments, the methodproduces a plurality of activated CAR-T cells, wherein at least 60% ofthe plurality of activated CAR-T cells expresses one or morecell-surface marker(s) of a stem memory T cell (T_(SCM)), therebyproducing a plurality of activated CAR stem memory T cells (T_(SCM)). Incertain embodiments, the method produces a plurality of activated CAR-Tcells, wherein at least 75% of the plurality of activated CAR-T cellsexpresses one or more cell-surface marker(s) of a stem memory T cell(T_(SCM)), thereby producing a plurality of activated CAR stem memory Tcells (T_(SCM)). In certain embodiments, the method produces a pluralityof activated CAR-T cells, wherein at least 80% of the plurality ofactivated CAR-T cells expresses one or more cell-surface marker(s) of astem memory T cell (T_(SCM)), thereby producing a plurality of activatedCAR stem memory T cells (T_(SCM)). In certain embodiments, the methodproduces a plurality of activated CAR-T cells, wherein at least 85% ofthe plurality of activated CAR-T cells expresses one or morecell-surface marker(s) of a stem memory T cell (T_(SCM)), therebyproducing a plurality of activated CAR stem memory T cells (T_(SCM)). Incertain embodiments, the method produces a plurality of activated CAR-Tcells, wherein at least 90% of the plurality of activated CAR-T cellsexpresses one or more cell-surface marker(s) of a stem memory T cell(T_(SCM)), thereby producing a plurality of activated CAR stem memory Tcells (T_(SCM)). In certain embodiments, the method produces a pluralityof activated CAR-T cells, wherein at least 95% of the plurality ofactivated CAR-T cells expresses one or more cell-surface marker(s) of astem memory T cell (T_(SCM)), thereby producing a plurality of activatedCAR stem memory T cells (T_(SCM)). In certain embodiments, thecell-surface markers comprise CD62L and CD45RA. In certain embodiments,the cell-surface markers of the activated CAR T_(SCM) comprise one ormore of CD62L, CD45RA, CD28, CCR7, CD127, CD45RO, CD95, CD95 and IL-2Rβ.In certain embodiments, the cell-surface markers of the activated CART_(SCM) comprise one or more of CD45RA, CD95, IL-2Rβ, CR7, and CD62L.The disclosure provides a method of producing a modified stem memory Tcell (T_(SCM)), comprising: (a) introducing into a primary human T cella composition comprising a chimeric antigen receptor (CAR) to produce aCAR-T cell and (b) contacting the CAR-T cell and a T-cell activatorcomposition comprising one or more of an anti-human CD3 monospecifictetrameric antibody complex, an anti-human CD28 monospecific tetramericantibody complex and an activation supplement to produce an activatedCAR-T cell, wherein the activated CAR-T cell expresses one or morecell-surface marker(s) of a stem memory T cell (T_(SCM)), therebyproducing a CAR-expressing stem memory T cell (T_(SCM)) (CAR-T_(SCM)).

The disclosure provides a method of producing a plurality of modifiedstem memory T cells (T_(SCM)), comprising: (a) introducing into aplurality of primary human T cells a composition comprising a chimericantigen receptor (CAR) to produce a plurality of CAR-T cells and (b)contacting the plurality of CAR-T cells and a T-cell activatorcomposition comprising one or more of an anti-human CD3 monospecifictetrameric antibody complex, an anti-human CD28 monospecific tetramericantibody complex and an activation supplement to produce a plurality ofactivated CAR-T cells, wherein at least 2%, 5%, 10%, 15%, 20%, 25%, 30%,35%, 40%, 45%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or anypercentage in between of the plurality of activated CAR-T cellsexpresses one or more cell-surface marker(s) of a stem memory T cell(T_(SCM)), thereby producing a plurality of activated CAR stem memory Tcells (T_(SCM)). In certain embodiments, the method produces a pluralityof activated CAR-T cells, wherein at least 25% of the plurality ofactivated CAR-T cells expresses one or more cell-surface marker(s) of astem memory T cell (T_(SCM)), thereby producing a plurality of activatedCAR stem memory T cells (T_(SCM)). In certain embodiments, the methodproduces a plurality of activated CAR-T cells, wherein at least 50% ofthe plurality of activated CAR-T cells expresses one or morecell-surface marker(s) of a stem memory T cell (T_(SCM)), therebyproducing a plurality of activated CAR stem memory T cells (T_(SCM)). Incertain embodiments, the method produces a plurality of activated CAR-Tcells, wherein at least 60% of the plurality of activated CAR-T cellsexpresses one or more cell-surface marker(s) of a stem memory T cell(T_(SCM)), thereby producing a plurality of activated CAR stem memory Tcells (T_(SCM)). In certain embodiments, the method produces a pluralityof activated CAR-T cells, wherein at least 75% of the plurality ofactivated CAR-T cells expresses one or more cell-surface marker(s) of astem memory T cell (T_(SCM)), thereby producing a plurality of activatedCAR stem memory T cells (T_(SCM)). In certain embodiments, the methodproduces a plurality of activated CAR-T cells, wherein at least 80% ofthe plurality of activated CAR-T cells expresses one or morecell-surface marker(s) of a stem memory T cell (T_(SCM)), therebyproducing a plurality of activated CAR stem memory T cells (T_(SCM)). Incertain embodiments, the method produces a plurality of activated CAR-Tcells, wherein at least 85% of the plurality of activated CAR-T cellsexpresses one or more cell-surface marker(s) of a stem memory T cell(T_(SCM)), thereby producing a plurality of activated CAR stem memory Tcells (T_(SCM)). In certain embodiments, the method produces a pluralityof activated CAR-T cells, wherein at least 90% of the plurality ofactivated CAR-T cells expresses one or more cell-surface marker(s) of astem memory T cell (T_(SCM)), thereby producing a plurality of activatedCAR stem memory T cells (T_(SCM)). In certain embodiments, the methodproduces a plurality of activated CAR-T cells, wherein at least 95% ofthe plurality of activated CAR-T cells expresses one or morecell-surface marker(s) of a stem memory T cell (T_(SCM)), therebyproducing a plurality of activated CAR stem memory T cells (T_(SCM)). Incertain embodiments, the cell-surface markers comprise CD62L and CD45RA.In certain embodiments, the cell-surface markers of the activated CART_(SCM) comprise one or more of CD62L, CD45RA, CD28, CCR7, CD127,CD45RO, CD95, CD95 and IL-2Rβ. In certain embodiments, the cell-surfacemarkers of the activated CAR T_(SCM) comprise one or more of CD45RA,CD95, IL-2Rβ, CR7, and CD62L.

In certain embodiments, this method may further comprise the step of:(c) contacting the activated CAR-T cell and a T-cell expansioncomposition comprising one or more of human serum albumin, recombinanthuman insulin, human transferrin, 2-Mercaptoethanol, Iscove's MDM, andan expansion supplement to produce a plurality of expanded CAR-T cells,wherein at least 2% of the plurality of expanded CAR-T cells expressesone or more cell-surface marker(s) of a stem memory T cell (T_(SCM))(CAR-T_(SCM)). In certain embodiments, the T-cell expansion compositionfurther comprises one or more of octanoic acid, nicotinamide,2,4,7,9-tetramethyl-5-decyn-4,7-diol (TMDD), diisopropyl adipate (DIPA),n-butyl-benzenesulfonamide, 1,2-benzenedicarboxylic acid,bis(2-methylpropyl) ester, palmitic acid, linoleic acid, oleic acid,stearic acid hydrazide, oleamide, a sterol and an alkane. In certainembodiments, the T-cell expansion composition comprises one or more ofoctanoic acid, palmitic acid, linoleic acid, oleic acid and a sterol(e.g. cholesterol). In certain embodiments, the T-cell expansioncomposition comprises one or more of octanoic acid at a concentration ofbetween 0.9 mg/kg to 90 mg/kg, inclusive of the endpoints; palmitic acidat a concentration of between 0.2 mg/kg to 20 mg/kg, inclusive of theendpoints; linoleic acid at a concentration of between 0.2 mg/kg to 20mg/kg, inclusive of the endpoints; oleic acid at a concentration of 0.2mg/kg to 20 mg/kg, inclusive of the endpoints; and a sterol at aconcentration of about 0.1 mg/kg to 10 mg/kg, inclusive of the endpoints(wherein mg/kg=parts per million). In certain embodiments, the T-cellexpansion composition comprises one or more of octanoic acid at aconcentration of about 9 mg/kg, palmitic acid at a concentration ofabout 2 mg/kg, linoleic acid at a concentration of about 2 mg/kg, oleicacid at a concentration of about 2 mg/kg, and a sterol at aconcentration of about 1 mg/kg (wherein mg/kg=parts per million). Incertain embodiments, the T-cell expansion composition comprises one ormore of octanoic acid at a concentration of 9.19 mg/kg, palmitic acid ata concentration of 1.86 mg/kg, linoleic acid at a concentration of about2.12 mg/kg, oleic acid at a concentration of about 2.13 mg/kg, and asterol at a concentration of about 1.01 mg/kg (wherein mg/kg=parts permillion). In certain embodiments, the T-cell expansion compositioncomprises octanoic acid at a concentration of 9.19 mg/kg, palmitic acidat a concentration of 1.86 mg/kg, linoleic acid at a concentration of2.12 mg/kg, oleic acid at a concentration of about 2.13 mg/kg, and asterol at a concentration of 1.01 mg/kg (wherein mg/kg=parts permillion). In certain embodiments, the T-cell expansion compositioncomprises one or more of octanoic acid at a concentration of between 6.4μmol/kg and 640 μmol/kg, inclusive of the endpoints; palmitic acid at aconcentration of between 0.7 μmol/kg and 70 μmol/kg, inclusive of theendpoints; linoleic acid at a concentration of between 0.75 μmol/kg and75 μmol/kg, inclusive of the endpoints; oleic acid at a concentration ofbetween 0.75 μmol/kg and 75 μmol/kg, inclusive of the endpoints; and asterol at a concentration of between 0.25 μmol/kg and 25 μmol/kg,inclusive of the endpoints. In certain embodiments, the T-cell expansioncomposition comprises one or more of octanoic acid at a concentration ofabout 64 μmol/kg, palmitic acid at a concentration of about 7 μmol/kg,linoleic acid at a concentration of about 7.5 μmol/kg, oleic acid at aconcentration of about 7.5 μmol/kg and a sterol at a concentration ofabout 2.5 μmol/kg. In certain embodiments, the T-cell expansioncomposition comprises one or more of octanoic acid at a concentration ofabout 63.75 μmol/kg, palmitic acid at a concentration of about 7.27μmol/kg, linoleic acid at a concentration of about 7.57 μmol/kg, oleicacid at a concentration of about 7.56 μmol/kg and a sterol at aconcentration of about 2.61 μmol/kg. In certain embodiments, the T-cellexpansion composition comprises octanoic acid at a concentration ofabout 63.75 μmol/kg, palmitic acid at a concentration of about 7.27μmol/kg, linoleic acid at a concentration of about 7.57 μmol/kg, oleicacid at a concentration of 7.56 μmol/kg and a sterol at a concentrationof 2.61 μmol/kg. In certain embodiments, at least 2%, 5%, 10%, 15%, 20%,25%, 30%, 35%, 40%, 45%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%,99% or any percentage in between of the plurality of expanded CAR-Tcells expresses cell-surface marker(s) of a stem memory T cell (T_(SCM))(CAR-T_(SCM)). In certain embodiments, the plurality of expanded CAR-Tcells may be enriched for CAR-T cells that express cell-surfacemarker(s) of a stem memory T cell (T_(SCM)) (CAR-T_(SCM)), and,therefore, following an enrichment step, the method may produce anenriched composition comprising at least 2%, 5%, 10%, 15%, 20%, 25%,30%, 35%, 40%, 45%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% orany percentage in between of CAR-T cells that express cell-surfacemarker(s) of a stem memory T cell (T_(SCM)) (CAR-T_(SCM)). In certainembodiments, the cell-surface markers comprise CD62L and CD45RA. Incertain embodiments, the cell-surface markers of the CAR-T_(SCM)comprise one or more of CD62L, CD45RA, CD28, CCR7, CD127, CD45RO, CD95,CD95 and IL-2Rβ. In certain embodiments, the cell-surface markers of theCAR-T_(SCM) comprise one or more of CD45RA, CD95, IL-2Rβ, CR7, andCD62L. In certain embodiments, the plurality of expanded CAR-T cellscomprises a naïve T-cell (CAR-T_(N)) and the cell-surface markers of theCAR-T_(N) comprise one or more of CD45RA, CCR7 and CD62L. In certainembodiments, the plurality of expanded CAR-T cells comprises a centralmemory T-cell (CAR-T_(CM)) and the cell-surface markers of theCAR-T_(CM) comprise one or more of CD45RO, CD95, IL-2Rβ, CCR7, andCD62L. In certain embodiments, the plurality of expanded CAR-T cellscomprises an effector memory T-cell (CAR-T_(EM)) and the cell-surfacemarkers of the CAR-T_(EM) comprise one or more of CD45RO, CD95, andIL-2Rβ. In certain embodiments, the plurality of expanded CAR-T cellscomprises an effector T-cell (CAR-T_(EFF)) and the cell-surface markersof the CAR-T_(EFF) comprise one or more of CD45RA, CD95, and IL-2Rβ.Additional cell-surface markers are described in Gattinoni et al. (NatMed. 2011 Sep. 18; 17(10): 1290-7; the contents of which areincorporated herein by reference in their entirety).

The disclosure provides a method of producing a modified stem memory Tcell (T_(SCM)), comprising: (a) introducing into a primary human T cella composition comprising a chimeric antigen receptor (CAR) to produce aCAR-T cell and (b) contacting the CAR-T cell and a T-cell activatorcomposition comprising one or more of an anti-human CD3 monospecifictetrameric antibody complex, an anti-human CD28 monospecific tetramericantibody complex and an activation supplement to produce an activatedCAR-T cell, wherein the activated CAR-T cell expresses one or morecell-surface marker(s) of a stem memory T cell (T_(SCM)), therebyproducing a CAR-expressing stem memory T cell (T_(SCM)) (CAR-T_(SCM)).The disclosure provides a method of producing a plurality of modifiedstem memory T cells (T_(SCM)), comprising: (a) introducing into aplurality of primary human T cells a composition comprising a chimericantigen receptor (CAR) to produce a plurality of CAR-T cells and (b)contacting the plurality of CAR-T cells and a T-cell activatorcomposition comprising one or more of an anti-human CD3 monospecifictetrameric antibody complex, an anti-human CD28 monospecific tetramericantibody complex and an activation supplement to produce a plurality ofactivated CAR-T cells, wherein at least 2%, 5%, 10%, 15%, 20%, 25%, 30%,35%, 40%, 45%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or anypercentage in between of the plurality of activated CAR-T cellsexpresses one or more cell-surface marker(s) of a stem memory T cell(T_(SCM)), thereby producing a plurality of activated CAR stem memory Tcells (T_(SCM)). In certain embodiments, the method produces a pluralityof activated CAR-T cells, wherein at least 25% of the plurality ofactivated CAR-T cells expresses one or more cell-surface marker(s) of astem memory T cell (T_(SCM)), thereby producing a plurality of activatedCAR stem memory T cells (T_(SCM)). In certain embodiments, the methodproduces a plurality of activated CAR-T cells, wherein at least 50% ofthe plurality of activated CAR-T cells expresses one or morecell-surface marker(s) of a stem memory T cell (T_(SCM)), therebyproducing a plurality of activated CAR stem memory T cells (T_(SCM)). Incertain embodiments, the method produces a plurality of activated CAR-Tcells, wherein at least 60% of the plurality of activated CAR-T cellsexpresses one or more cell-surface marker(s) of a stem memory T cell(T_(SCM)), thereby producing a plurality of activated CAR stem memory Tcells (T_(SCM)). In certain embodiments, the method produces a pluralityof activated CAR-T cells, wherein at least 75% of the plurality ofactivated CAR-T cells expresses one or more cell-surface marker(s) of astem memory T cell (T_(SCM)), thereby producing a plurality of activatedCAR stem memory T cells (T_(SCM)). In certain embodiments, the methodproduces a plurality of activated CAR-T cells, wherein at least 80% ofthe plurality of activated CAR-T cells expresses one or morecell-surface marker(s) of a stem memory T cell (T_(SCM)), therebyproducing a plurality of activated CAR stem memory T cells (T_(SCM)). Incertain embodiments, the method produces a plurality of activated CAR-Tcells, wherein at least 85% of the plurality of activated CAR-T cellsexpresses one or more cell-surface marker(s) of a stem memory T cell(T_(SCM)), thereby producing a plurality of activated CAR stem memory Tcells (T_(SCM)). In certain embodiments, the method produces a pluralityof activated CAR-T cells, wherein at least 90% of the plurality ofactivated CAR-T cells expresses one or more cell-surface marker(s) of astem memory T cell (T_(SCM)), thereby producing a plurality of activatedCAR stem memory T cells (T_(SCM)). In certain embodiments, the methodproduces a plurality of activated CAR-T cells, wherein at least 95% ofthe plurality of activated CAR-T cells expresses one or morecell-surface marker(s) of a stem memory T cell (T_(SCM)), therebyproducing a plurality of activated CAR stem memory T cells (T_(SCM)). Incertain embodiments, the cell-surface markers comprise CD62L and CD45RA.In certain embodiments, the cell-surface markers of the activated CART_(SCM) comprise one or more of CD62L, CD45RA, CD28, CCR7, CD127,CD45RO, CD95, CD95 and IL-2Rβ. In certain embodiments, the cell-surfacemarkers of the activated CAR T_(SCM) comprise one or more of CD45RA,CD95, IL-2Rβ, CR7, and CD62L.

In certain embodiments of the methods of the disclosure, the pluralityof expanded CAR-T cells comprises a naïve T-cell (CAR-T_(N)) and thecell-surface markers of the CAR-T_(N) comprise one or more of CD45RA,CCR7 and CD62L. In certain embodiments, the plurality of expanded CAR-Tcells comprises a central memory T-cell (CAR-T_(CM)) and thecell-surface markers of the CAR-T_(CM) comprise one or more of CD45RO,CD95, CCR7, and CD62L. In certain embodiments, the plurality of expandedCAR-T cells comprises an effector memory T-cell (CAR-T_(EM)) and thecell-surface markers of the CAR-T_(EM) comprise one or more of CD45RO,CD95, and IL-2Rβ. In certain embodiments, the plurality of expandedCAR-T cells comprises an effector T-cell (CAR-T_(EFF)) and thecell-surface markers of the CAR-T_(EFF) comprise one or more of CD45RA,CD95, and IL-2Rβ.

In certain embodiments of the methods of the disclosure, a transposoncomprises a chimeric antigen receptor (CAR) of the disclosure. Thetransposon may be a plasmid DNA transposon with a sequence encoding theCAR flanked by two cis-regulatory insulator elements. In certainpreferred embodiments, the transposon is a piggyBac transposon. Incertain embodiments, a step introducing a composition comprising achimeric antigen receptor (CAR) of the disclosure may further acomposition comprising an mRNA sequence encoding a transposase. Incertain preferred embodiments, the transposase is a Super piggyBac™(SPB) transposase.

In certain embodiments, a transposon of the disclosure may furthercomprise a selection gene. When a transposon of the disclosure comprisesa selection gene, the T-cell expansion composition of the methods of thedisclosure may further comprise a selection agent to simultaneouslyselect and expand an activated or modified T cell of the disclosure.

In certain embodiments a CAR of the disclosure may be a CARTyrin. Incertain embodiments, the CAR comprises one or more VHH sequence(s). Incertain embodiments, the CAR is a VCAR.

In certain embodiments of the methods of producing a modified T_(SCM) ofthe disclosure, the introducing step may comprise an electroporation ora nucleofection. When the introducing step comprises a nucleofection,the nucleofection may comprise the steps of: (a) contacting a transposoncomposition, a transposase composition, and a composition comprising aplurality of primary human T cells in a cuvette; (b) applying one ormore electrical pulses to the cuvette, and (c) incubating thecomposition comprising the plurality of primary human T cells in acomposition comprising a T-cell expansion composition comprising one ormore of human serum albumin, recombinant human insulin, humantransferrin, 2-Mercaptoethanol, Iscove's MDM, and an expansionsupplement at 37° C. In certain embodiments, the T-cell expansioncomposition further comprises one or more of octanoic acid,nicotinamide, 2,4,7,9-tetramethyl-5-decyn-4,7-diol (TMDD), diisopropyladipate (DIPA), n-butyl-benzenesulfonamide, 1,2-benzenedicarboxylicacid, bis(2-methylpropyl) ester, palmitic acid, linoleic acid, oleicacid, stearic acid hydrazide, oleamide, a sterol and an alkane. Incertain embodiments, the T-cell expansion composition comprises one ormore of octanoic acid, palmitic acid, linoleic acid, oleic acid and asterol (e.g. cholesterol). In certain embodiments, the T-cell expansioncomposition comprises one or more of octanoic acid at a concentration ofbetween 0.9 mg/kg to 90 mg/kg, inclusive of the endpoints; palmitic acidat a concentration of between 0.2 mg/kg to 20 mg/kg, inclusive of theendpoints; linoleic acid at a concentration of between 0.2 mg/kg to 20mg/kg, inclusive of the endpoints; oleic acid at a concentration of 0.2mg/kg to 20 mg/kg, inclusive of the endpoints; and a sterol at aconcentration of about 0.1 mg/kg to 10 mg/kg, inclusive of the endpoints(wherein mg/kg=parts per million). In certain embodiments, the T-cellexpansion composition comprises one or more of octanoic acid at aconcentration of about 9 mg/kg, palmitic acid at a concentration ofabout 2 mg/kg, linoleic acid at a concentration of about 2 mg/kg, oleicacid at a concentration of about 2 mg/kg, and a sterol at aconcentration of about 1 mg/kg (wherein mg/kg=parts per million). Incertain embodiments, the T-cell expansion composition comprises one ormore of octanoic acid at a concentration of 9.19 mg/kg, palmitic acid ata concentration of 1.86 mg/kg, linoleic acid at a concentration of about2.12 mg/kg, oleic acid at a concentration of about 2.13 mg/kg, and asterol at a concentration of about 1.01 mg/kg (wherein mg/kg=parts permillion). In certain embodiments, the T-cell expansion compositioncomprises octanoic acid at a concentration of 9.19 mg/kg, palmitic acidat a concentration of 1.86 mg/kg, linoleic acid at a concentration of2.12 mg/kg, oleic acid at a concentration of about 2.13 mg/kg, and asterol at a concentration of 1.01 mg/kg (wherein mg/kg=parts permillion). In certain embodiments, the T-cell expansion compositioncomprises one or more of octanoic acid at a concentration of between 6.4μmol/kg and 640 μmol/kg, inclusive of the endpoints; palmitic acid at aconcentration of between 0.7 μmol/kg and 70 μmol/kg, inclusive of theendpoints; linoleic acid at a concentration of between 0.75 μmol/kg and75 μmol/kg, inclusive of the endpoints; oleic acid at a concentration ofbetween 0.75 μmol/kg and 75 μmol/kg, inclusive of the endpoints; and asterol at a concentration of between 0.25 μmol/kg and 25 μmol/kg,inclusive of the endpoints. In certain embodiments, the T-cell expansioncomposition comprises one or more of octanoic acid at a concentration ofabout 64 μmol/kg, palmitic acid at a concentration of about 7 μmol/kg,linoleic acid at a concentration of about 7.5 μmol/kg, oleic acid at aconcentration of about 7.5 μmol/kg and a sterol at a concentration ofabout 2.5 μmol/kg. In certain embodiments, the T-cell expansioncomposition comprises one or more of octanoic acid at a concentration ofabout 63.75 μmol/kg, palmitic acid at a concentration of about 7.27μmol/kg, linoleic acid at a concentration of about 7.57 μmol/kg, oleicacid at a concentration of about 7.56 μmol/kg and a sterol at aconcentration of about 2.61 μmol/kg. In certain embodiments, the T-cellexpansion composition comprises octanoic acid at a concentration ofabout 63.75 μmol/kg, palmitic acid at a concentration of about 7.27μmol/kg, linoleic acid at a concentration of about 7.57 μmol/kg, oleicacid at a concentration of 7.56 μmol/kg and a sterol at a concentrationof 2.61 μmol/kg. In certain embodiments of the nucleofection, thetransposon composition is a 0.5 μg/μl solution comprising nuclease freewater and the cuvette comprises 2 μl of the transposon composition toyield 1 μg of transposon. The transposon composition may comprise apiggyBac transposon. The transposon composition may comprise a SleepingBeauty transposon. In certain embodiments of the nucleofection, thetransposase composition comprises 5 μg of transposase. The transposasecomposition may comprise a hyperactive piggyBac™ or Super piggyBac™(SPB) transposase. The transposase composition may comprise ahyperactive Sleeping Beauty (SB100X) transposase. In certainembodiments, the transposon may comprise a Helraiser transposon and thetransposase composition may comprise a Helitron transposase. In certainembodiments, the transposon may comprise a Tol2 transposon and thetransposase composition comprises a Tol2 transposase.

In certain embodiments of the methods of the disclosure, including thoseembodiments wherein the introducing step comprises a nucleofection or anelectroporation, the nucleofection comprises contacting a firsttransposon composition and a first transposase composition and acomposition comprising a plurality of primary human T cells in acuvette. In certain embodiments of the methods of the disclosure,including those embodiments wherein the introducing step comprises anucleofection or an electroporation, the nucleofection comprisescontacting a first transposon composition, a second transposoncomposition, a first transposase composition and a compositioncomprising a plurality of primary human T cells in a cuvette. In certainembodiments of the methods of the disclosure, including thoseembodiments wherein the introducing step comprises a nucleofection or anelectroporation, the nucleofection comprises contacting a firsttransposon composition, a second transposon composition, a firsttransposase composition, a second transposase composition and acomposition comprising a plurality of primary human T cells in acuvette. In certain embodiments, the first transposon comprises asequence encoding an antigen receptor. In certain embodiments, thesecond transposon comprises a sequence encoding a therapeutic protein.In certain embodiments, the first transposon composition and the secondtransposon composition are identical. In certain embodiments, the firsttransposon composition and the second transposon composition are notidentical. In certain embodiments, the first transposase mobilizes thefirst transposon composition and the second transposon composition. Incertain embodiments, the first transposase mobilizes the firsttransposon composition but not the second transposon composition. Incertain embodiments, the second transposase mobilizes the secondtransposon composition but not the first transposon composition. Incertain embodiments, the first transposase mobilizes the firsttransposon composition and the second transposase mobilizes the secondtransposon composition. In certain embodiments, the first transposoncomposition or the second transposon composition comprises a sequenceencoding an antigen receptor. In certain embodiments, the firsttransposon composition or the second transposon composition comprises asequence encoding a therapeutic protein. In certain embodiments, thefirst transposon composition comprises a sequence encoding an antigenreceptor and the second transposon composition comprises a sequenceencoding a therapeutic protein. In certain embodiments, the therapeuticprotein is a secreted or secretable protein. In certain embodiments ofthe methods of the disclosure, including those embodiments wherein theintroducing step comprises a nucleofection or an electroporation, thenucleofection comprises contacting a transposon composition, a firsttransposase composition, a second transposase composition and acomposition comprising a plurality of primary human T cells in acuvette. In certain embodiments, the transposon composition comprises asequence encoding the antigen receptor. In certain embodiments, thetransposon composition comprises a sequence encoding the therapeuticprotein. In certain embodiments of the methods of the disclosure,including those embodiments wherein the introducing step comprises anucleofection or an electroporation, the nucleofection further comprisescontacting a composition capable of inducing homologous recombination ata specific site in the genome with a composition comprising a pluralityof primary human T cells in a cuvette. In certain embodiments, thecomposition capable of inducing homologous recombination comprises anexogenous donor molecule. In certain embodiments, the exogenous donormolecule comprises a sequence encoding the antigen receptor and thetransposon comprises a sequence encoding the therapeutic protein. Incertain embodiments, the exogenous donor molecule comprises a sequenceencoding the therapeutic protein and the transposon comprises a sequenceencoding the antigen receptor. In certain embodiments, the compositioncomprising the transposon, the composition comprising the transposaseand the composition capable of inducing homologous recombination at aspecific site in the genome are contacted with the compositioncomprising a plurality of primary human T cells simultaneously. Incertain embodiments, the composition comprising the transposon and thecomposition comprising the transposase are contacted with thecomposition comprising a plurality of primary human T cells first, andthe composition capable of inducing homologous recombination at aspecific site in the genome is contacted with the composition comprisinga plurality of primary human T cells second. In certain embodiments, thecomposition capable of inducing homologous recombination at a specificsite in the genome is contacted with the composition comprising aplurality of primary human T cells first and the composition comprisingthe transposon and the composition comprising the transposase arecontacted with the composition comprising a plurality of primary human Tcells second. In certain embodiments of the methods of producing amodified T_(SCM) of the disclosure, the composition comprising primaryhuman T cells comprises a buffer that maintains or enhances a level ofcell viability and/or a stem-like phenotype of the primary human Tcells. In certain embodiments, the buffer maintains or enhances a levelof cell viability and/or a stem-like phenotype of the primary human Tcells prior to the nucleofection. In certain embodiments, the buffermaintains or enhances a level of cell viability and/or a stem-likephenotype of the primary human T cells during the nucleofection. Incertain embodiments, the buffer maintains or enhances a level of cellviability and/or a stem-like phenotype of the primary human T cellsfollowing the nucleofection. In certain embodiments, the buffercomprises a P3 primary cell solution (Lonza). In certain embodiments,the buffer comprises one or more of KCl, MgCl₂, ClNa, Glucose andCa(NO₃)₂ in any absolute or relative abundance or concentration, and,optionally, the buffer further comprises a supplement selected from thegroup consisting of HEPES, Tris/HCl, and a phosphate buffer. In certainembodiments, the buffer comprises 5 mM KCl, 15 mM MgCl₂, 90 mM ClNa, 10mM Glucose and 0.4 mM Ca(NO₃)₂. In certain embodiments, the buffercomprises 5 mM KCl, 15 mM MgCl₂, 90 mM ClNa, 10 mM Glucose and 0.4 mMCa(NO₃)₂ and a supplement comprising 20 mM HEPES and 75 mM Tris/HCl. Incertain embodiments, the buffer comprises 5 mM KCl, 15 mM MgCl₂, 90 mMClNa, 10 mM Glucose and 0.4 mM Ca(NO₃)₂ and a supplement comprising 40mM Na₂HPO₄/NaH₂PO₄ at pH 7.2. In certain embodiments, the compositioncomprising primary human T cells comprises 100 μl of the buffer andbetween 5×10⁶ and 25×10⁶ cells.

In certain embodiments of the methods of producing a modified T_(SCM) ofthe disclosure, the composition comprising primary human T cells isdepleted of cells expressing CD14, CD56, and/or CD19. In certainembodiments, the composition comprising primary human T cells comprises100 μl of the buffer and between 5×10⁶ and 25×10⁶ cells.

As used herein, the terms “supplemented T-cell expansion composition” or“T-cell expansion composition” may be used interchangeably with a mediacomprising one or more of human serum albumin, recombinant humaninsulin, human transferrin, 2-Mercaptoethanol, Iscove's MDM, and anexpansion supplement at 37° C. Alternatively, or in addition, the terms“supplemented T-cell expansion composition” or “T-cell expansioncomposition” may be used interchangeably with a media comprising one ormore of phosphorus, an octanoic fatty acid, a palmitic fatty acid, alinoleic fatty acid and an oleic acid. In certain embodiments, the mediacomprises an amount of phosphorus that is 10-fold higher than may befound in, for example, Iscove's Modified Dulbecco's Medium ((IMDM);available at ThermoFisher Scientific as Catalog number 12440053).

As used herein, the terms “supplemented T-cell expansion composition” or“T-cell expansion composition” may be used interchangeably with a mediacomprising one or more of human serum albumin, recombinant humaninsulin, human transferrin, 2-Mercaptoethanol, Iscove's MDM, and anexpansion supplement at 37° C. Alternatively, or in addition, the terms“supplemented T-cell expansion composition” or “T-cell expansioncomposition” may be used interchangeably with a media comprising one ormore of the following elements: boron, sodium, magnesium, phosphorus,potassium, and calcium. In certain embodiments, the terms “supplementedT-cell expansion composition” or “T-cell expansion composition” may beused interchangeably with a media comprising one or more of thefollowing elements present in the corresponding average concentrations:boron at 3.7 mg/L, sodium at 3000 mg/L, magnesium at 18 mg/L, phosphorusat 29 mg/L, potassium at 15 mg/L and calcium at 4 mg/L.

As used herein, the terms “supplemented T-cell expansion composition” or“T-cell expansion composition” may be used interchangeably with a mediacomprising one or more of human serum albumin, recombinant humaninsulin, human transferrin, 2-Mercaptoethanol, Iscove's MDM, and anexpansion supplement at 37° C. Alternatively, or in addition, the terms“supplemented T-cell expansion composition” or “T-cell expansioncomposition” may be used interchangeably with a media comprising one ormore of the following components: octanoic acid (CAS No. 124-07-2),nicotinamide (CAS No. 98-92-0), 2,4,7,9-tetramethyl-5-decyn-4,7-diol(TMDD) (CAS No. 126-86-3), diisopropyl adipate (DIPA) (CAS No.6938-94-9), n-butyl-benzenesulfonamide (CAS No. 3622-84-2),1,2-benzenedicarboxylic acid, bis(2-methylpropyl) ester (CAS No.84-69-5), palmitic acid (CAS No. 57-10-3), linoleic acid (CAS No.60-33-3), oleic acid (CAS No. 112-80-1), stearic acid hydrazide (CAS No.4130-54-5), oleamide (CAS No. 3322-62-1), sterol (e.g., cholesterol)(CAS No. 57-88-5), and alkanes (e.g., nonadecane) (CAS No. 629-92-5). Incertain embodiments, the terms “supplemented T-cell expansioncomposition” or “T-cell expansion composition” may be usedinterchangeably with a media comprising one or more of the followingcomponents: octanoic acid (CAS No. 124-07-2), nicotinamide (CAS No.98-92-0), 2,4,7,9-tetramethyl-5-decyn-4,7-diol (TMDD) (CAS No.126-86-3), diisopropyl adipate (DIPA) (CAS No. 6938-94-9),n-butyl-benzenesulfonamide (CAS No. 3622-84-2), 1,2-benzenedicarboxylicacid, bis(2-methylpropyl) ester (CAS No. 84-69-5), palmitic acid (CASNo. 57-10-3), linoleic acid (CAS No. 60-33-3), oleic acid (CAS No.112-80-1), stearic acid hydrazide (CAS No. 4130-54-5), oleamide (CAS No.3322-62-1), sterol (e.g., cholesterol) (CAS No. 57-88-5), alkanes (e.g.,nonadecane) (CAS No. 629-92-5), and phenol red (CAS No. 143-74-8). Incertain embodiments, the terms “supplemented T-cell expansioncomposition” or “T-cell expansion composition” may be usedinterchangeably with a media comprising one or more of the followingcomponents: octanoic acid (CAS No. 124-07-2), nicotinamide (CAS No.98-92-0), 2,4,7,9-tetramethyl-5-decyn-4,7-diol (TMDD) (CAS No.126-86-3), diisopropyl adipate (DIPA) (CAS No. 6938-94-9),n-butyl-benzenesulfonamide (CAS No. 3622-84-2), 1,2-benzenedicarboxylicacid, bis(2-methylpropyl) ester (CAS No. 84-69-5), palmitic acid (CASNo. 57-10-3), linoleic acid (CAS No. 60-33-3), oleic acid (CAS No.112-80-1), stearic acid hydrazide (CAS No. 4130-54-5), oleamide (CAS No.3322-62-1), phenol red (CAS No. 143-74-8) and lanolin alcohol.

As used herein, the terms “supplemented T-cell expansion composition” or“T-cell expansion composition” may be used interchangeably with a mediacomprising one or more of human serum albumin, recombinant humaninsulin, human transferrin, 2-Mercaptoethanol, Iscove's MDM, and anexpansion supplement at 37° C. Alternatively, or in addition, the terms“supplemented T-cell expansion composition” or “T-cell expansioncomposition” may be used interchangeably with a media comprising one ormore of the following ions: sodium, ammonium, potassium, magnesium,calcium, chloride, sulfate and phosphate.

As used herein, the terms “supplemented T-cell expansion composition” or“T-cell expansion composition” may be used interchangeably with a mediacomprising one or more of human serum albumin, recombinant humaninsulin, human transferrin, 2-Mercaptoethanol, Iscove's MDM, and anexpansion supplement at 37° C. Alternatively, or in addition, the terms“supplemented T-cell expansion composition” or “T-cell expansioncomposition” may be used interchangeably with a media comprising one ormore of the following free amino acids: histidine, asparagine, serine,glutamate, arginine, glycine, aspartic acid, glutamic acid, threonine,alanine, proline, cysteine, lysine, tyrosine, methionine, valine,isoleucine, leucine, phenylalanine and tryptophan. In certainembodiments, the terms “supplemented T-cell expansion composition” or“T-cell expansion composition” may be used interchangeably with a mediacomprising one or more of the following free amino acids in thecorresponding average mole percentages: histidine (about 1%), asparagine(about 0.5%), serine (about 1.5%), glutamine (about 67%), arginine(about 1.5%), glycine (about 1.5%), aspartic acid (about 1%), glutamicacid (about 2%), threonine (about 2%), alanine (about 1%), proline(about 1.5%), cysteine (about 1.5%), lysine (about 3%), tyrosine (about1.5%), methionine (about 1%), valine (about 3.5%), isoleucine (about3%), leucine (about 3.5%), phenylalanine (about 1.5%) and tryptophan(about 0.5%). In certain embodiments, the terms “supplemented T-cellexpansion composition” or “T-cell expansion composition” may be usedinterchangeably with a media comprising one or more of the followingfree amino acids in the corresponding average mole percentages:histidine (about 0.78%), asparagine (about 0.4%), serine (about 1.6%),glutamine (about 67.01%), arginine (about 1.67%), glycine (about 1.72%),aspartic acid (about 1.00%), glutamic acid (about 1.93%), threonine(about 2.38%), alanine (about 1.11%), proline (about 1.49%), cysteine(about 1.65%), lysine (about 2.84%), tyrosine (about 1.62%), methionine(about 0.85%), valine (about 3.45%), isoleucine (about 3.14%), leucine(about 3.3%), phenylalanine (about 1.64%) and tryptophan (about 0.37%).

As used herein, the terms “supplemented T-cell expansion composition” or“T-cell expansion composition” may be used interchangeably with a mediacomprising one or more of octanoic acid, palmitic acid, linoleic acid,oleic acid and a sterol (e.g. cholesterol). In certain embodiments, theterms “supplemented T-cell expansion composition” or “T-cell expansioncomposition” may be used interchangeably with a media comprising one ormore of octanoic acid at a concentration of between 0.9 mg/kg to 90mg/kg, inclusive of the endpoints; palmitic acid at a concentration ofbetween 0.2 mg/kg to 20 mg/kg, inclusive of the endpoints; linoleic acidat a concentration of between 0.2 mg/kg to 20 mg/kg, inclusive of theendpoints; oleic acid at a concentration of 0.2 mg/kg to 20 mg/kg,inclusive of the endpoints; and a sterol at a concentration of about 0.1mg/kg to 10 mg/kg, inclusive of the endpoints (wherein mg/kg=parts permillion). In certain embodiments, the terms “supplemented T-cellexpansion composition” or “T-cell expansion composition” may be usedinterchangeably with a media comprising one or more of octanoic acid ata concentration of about 9 mg/kg, palmitic acid at a concentration ofabout 2 mg/kg, linoleic acid at a concentration of about 2 mg/kg, oleicacid at a concentration of about 2 mg/kg, and a sterol at aconcentration of about 1 mg/kg (wherein mg/kg=parts per million).). Incertain embodiments, the terms “supplemented T-cell expansioncomposition” or “T-cell expansion composition” may be usedinterchangeably with a media comprising one or more of octanoic acid ata concentration of 9.19 mg/kg, palmitic acid at a concentration of 1.86mg/kg, linoleic acid at a concentration of about 2.12 mg/kg, oleic acidat a concentration of about 2.13 mg/kg, and a sterol at a concentrationof about 1.01 mg/kg (wherein mg/kg=parts per million). In certainembodiments, the terms “supplemented T-cell expansion composition” or“T-cell expansion composition” may be used interchangeably with a mediacomprising one or more of octanoic acid at a concentration of 9.19mg/kg, palmitic acid at a concentration of 1.86 mg/kg, linoleic acid ata concentration of 2.12 mg/kg, oleic acid at a concentration of about2.13 mg/kg, and a sterol at a concentration of 1.01 mg/kg (whereinmg/kg=parts per million). In certain embodiments, the terms“supplemented T-cell expansion composition” or “T-cell expansioncomposition” may be used interchangeably with a media comprising one ormore of octanoic acid at a concentration of between 6.4 μmol/kg and 640μmol/kg, inclusive of the endpoints; palmitic acid at a concentration ofbetween 0.7 μmol/kg and 70 μmol/kg, inclusive of the endpoints; linoleicacid at a concentration of between 0.75 μmol/kg and 75 μmol/kg,inclusive of the endpoints; oleic acid at a concentration of between0.75 μmol/kg and 75 μmol/kg, inclusive of the endpoints; and a sterol ata concentration of between 0.25 μmol/kg and 25 μmol/kg, inclusive of theendpoints. In certain embodiments, the terms “supplemented T-cellexpansion composition” or “T-cell expansion composition” may be usedinterchangeably with a media comprising one or more of octanoic acid ata concentration of about 64 μmol/kg, palmitic acid at a concentration ofabout 7 μmol/kg, linoleic acid at a concentration of about 7.5 μmol/kg,oleic acid at a concentration of about 7.5 μmol/kg and a sterol at aconcentration of about 2.5 μmol/kg. In certain embodiments, the terms“supplemented T-cell expansion composition” or “T-cell expansioncomposition” may be used interchangeably with a media comprising one ormore of octanoic acid at a concentration of about 63.75 μmol/kg,palmitic acid at a concentration of about 7.27 μmol/kg, linoleic acid ata concentration of about 7.57 μmol/kg, oleic acid at a concentration ofabout 7.56 μmol/kg and a sterol at a concentration of about 2.61μmol/kg. In certain embodiments, the terms “supplemented T-cellexpansion composition” or “T-cell expansion composition” may be usedinterchangeably with a media comprising one or more of octanoic acid ata concentration of about 63.75 μmol/kg, palmitic acid at a concentrationof about 7.27 μmol/kg, linoleic acid at a concentration of about 7.57μmol/kg, oleic acid at a concentration of 7.56 μmol/kg and a sterol at aconcentration of 2.61 μmol/kg.

As used herein, the term “P3 buffer” may be used interchangeably with abuffer comprising one or more of KCl, MgCl₂, ClNa, Glucose and Ca(NO₃)₂in any absolute or relative abundance or concentration, and, optionally,the further comprising a supplement selected from the group consistingof HEPES, Tris/HCl, and a phosphate buffer. The term “P3 buffer” may beused interchangeably with a buffer comprising 5 mM KCl, 15 mM MgCl₂, 90mM ClNa, 10 mM Glucose and 0.4 mM Ca(NO₃)₂, and, optionally, the furthercomprising a supplement selected from the group consisting of HEPES,Tris/HCl, and a phosphate buffer. The term “P3 buffer” may be usedinterchangeably with a buffer comprising 5 mM KCl, 15 mM MgCl₂, 90 mMClNa, 10 mM Glucose and 0.4 mM Ca(NO₃)₂ and a supplement comprising 20mM HEPES and 75 mM Tris/HCl. The term “P3 buffer” may be usedinterchangeably with a buffer comprising 5 mM KCl, 15 mM MgCl₂, 90 mMClNa, 10 mM Glucose and 0.4 mM Ca(NO₃)₂ and a supplement comprising 40mM Na₂HPO₄/NaH₂PO₄ at pH 7.2.

As used herein, the terms “supplemented RPMI-1640 media” or “T-cellconditioned media (TCCM)” may be used interchangeably with a mediacomprising one or more of water, fetal bovine serum, HEPES, sodiumpyruvate, one or more non-essential amino acids, a phenol red indicator,calcium nitrate, magnesium sulfate, potassium chloride, sodiumbicarbonate, sodium chloride, sodium phosphate dibasic (anhydrous),L-Alanyl-L-Glutamine, L-Arginine, L-Asparagine (anhydrous), L-Asparticacid, L-Cysteine 2HCl, L-Glutamic acid, Glycine, L-Histidine,Hydroxy-L-Proline, L-Isoleucine, L-Leucine, L-Lysine HCl, L-Methionine,L-Phenylalanine, L-Proline, L-Serine, L-Threonine, L-Tryptophan,L-Tyrosine 2Na 2H₂O, L-Valine, D-Biotin, choline chloride, folic acid,Myo-Inositol, niacinamide, p-Aminobenzoic acid, D-Panthothenic acid(hemicalcium), pyridoxine HCl, riboflavin, thiamine HCl, vitamin B12,D-Glucose, Glutathione (reduced), L-Glutamine and 2-Mercaptoethanol inany absolute or relative abundance or concentration. The terms“supplemented RPMI-1640 media” or “T-cell conditioned media (TCCM)” maybe used interchangeably with a media comprising water, fetal bovineserum, HEPES, sodium pyruvate, one or more non-essential amino acids, aphenol red indicator, calcium nitrate, magnesium sulfate, potassiumchloride, sodium bicarbonate, sodium chloride, sodium phosphate dibasic(anhydrous), L-Alanyl-L-Glutamine, L-Arginine, L-Asparagine (anhydrous),L-Aspartic acid, L-Cysteine 2HCl, L-Glutamic acid, Glycine, L-Histidine,Hydroxy-L-Proline, L-Isoleucine, L-Leucine, L-Lysine HCl, L-Methionine,L-Phenylalanine, L-Proline, L-Serine, L-Threonine, L-Tryptophan,L-Tyrosine 2Na 2H₂O, L-Valine, D-Biotin, choline chloride, folic acid,Myo-Inositol, niacinamide, p-Aminobenzoic acid, D-Panthothenic acid(hemicalcium), pyridoxine HCl, riboflavin, thiamine HCl, vitamin B12,D-Glucose, Glutathione (reduced), L-Glutamine and 2-Mercaptoethanol inany absolute or relative abundance or concentration.

As used herein, the terms “supplemented AIM-V” or “supplemented AIMV”media may be used interchangeably with a media comprising one or more ofwater, human serum albumin, streptomycin sulfate, gentamicin, fetalbovine serum, HEPES, sodium pyruvate, one or more non-essential aminoacids, a phenol red indicator, calcium nitrate, magnesium sulfate,potassium chloride, sodium bicarbonate, sodium chloride, sodiumphosphate dibasic (anhydrous), L-Alanyl-L-Glutamine, L-Arginine,L-Asparagine (anhydrous), L-Aspartic acid, L-Cysteine 2HCl, L-Glutamicacid, Glycine, L-Histidine, Hydroxy-L-Proline, L-Isoleucine, L-Leucine,L-Lysine HCl, L-Methionine, L-Phenylalanine, L-Proline, L-Serine,L-Threonine, L-Tryptophan, L-Tyrosine 2Na 2H₂O, L-Valine, D-Biotin,choline chloride, folic acid, Myo-Inositol, niacinamide, p-Aminobenzoicacid, D-Panthothenic acid (hemicalcium), pyridoxine HCl, riboflavin,thiamine HCl, vitamin B12, D-Glucose, glutathione (reduced), L-Glutamineand 2-Mercaptoethanol in any absolute or relative abundance orconcentration. The terms “supplemented AIM-V” or “supplemented AIMV”media may be used interchangeably with a media comprising water, humanserum albumin, streptomycin sulfate, gentamicin, fetal bovine serum,HEPES, sodium pyruvate, one or more non-essential amino acids, a phenolred indicator, calcium nitrate, magnesium sulfate, potassium chloride,sodium bicarbonate, sodium chloride, sodium phosphate dibasic(anhydrous), L-Alanyl-L-Glutamine, L-Arginine, L-Asparagine (anhydrous),L-Aspartic acid, L-Cysteine 2HCl, L-Glutamic acid, Glycine, L-Histidine,Hydroxy-L-Proline, L-Isoleucine, L-Leucine, L-Lysine HCl, L-Methionine,L-Phenylalanine, L-Proline, L-Serine, L-Threonine, L-Tryptophan,L-Tyrosine 2Na 2H₂O, L-Valine, D-Biotin, choline chloride, folic acid,Myo-Inositol, niacinamide, p-Aminobenzoic acid, D-Panthothenic acid(hemicalcium), pyridoxine HCl, riboflavin, thiamine HCl, vitamin B12,D-Glucose, glutathione (reduced), L-Glutamine and 2-Mercaptoethanol inany absolute or relative abundance or concentration.

As used herein, the term “ImmunoCult™ medium” may be usedinterchangeably with a medium comprising one or more of water, humanserum albumin, recombinant human insulin, human transferrin,2-Mercaptoethanol, L-Glutamine, phenol red, glycine, L-Alanine,L-Arginine hydrochloride, L-Asparagine, L-Aspartic acid, L-Cysteine2HCl, L-Glutamic acid, L-Glutamine, L-Histidine hydrochloride H₂O,L-Isoleucine, L-Leucine, L-Lysine hydrochloride, L-Methionine,L-Phenylalanine, L-Proline, L-Serine, L-Threonine, L-Tryptophan,L-Tyrosine disodium salt, L-Valine, biotin, choline chloride, D-Calciumpantothenate, folic acid, niacinamide, pyridoxal hydrochloride,riboflavin, thiamine hydrochloride, vitamin B12, i-Inositol, calciumchloride (anhydrous), magnesium sulfate (Anhydrous), potassium chloride,potassium nitrate, sodium bicarbonate, sodium chloride, sodium phosphatemonobasic, sodium selenite, D-Glucose, HEPES and Sodium pyruvate in anyabsolute or relative abundance or concentration. The term “ImmunoCult™medium” may be used interchangeably with a medium comprising water,human serum albumin, recombinant human insulin, human transferrin,2-Mercaptoethanol, L-Glutamine, phenol red, glycine, L-Alanine,L-Arginine hydrochloride, L-Asparagine, L-Aspartic acid, L-Cysteine2HCl, L-Glutamic acid, L-Glutamine, L-Histidine hydrochloride H₂O,L-Isoleucine, L-Leucine, L-Lysine hydrochloride, L-Methionine,L-Phenylalanine, L-Proline, L-Serine, L-Threonine, L-Tryptophan,L-Tyrosine disodium salt, L-Valine, biotin, choline chloride, D-Calciumpantothenate, folic acid, niacinamide, pyridoxal hydrochloride,riboflavin, thiamine hydrochloride, vitamin B12, i-Inositol, calciumchloride (anhydrous), magnesium sulfate (Anhydrous), potassium chloride,potassium nitrate, sodium bicarbonate, sodium chloride, sodium phosphatemonobasic, sodium selenite, D-Glucose, HEPES and Sodium pyruvate in anyabsolute or relative abundance or concentration.

Modified T-cells of the disclosure, including modified T_(SCM) and/orT_(CM) of the disclosure, may be incubated, cultured, grown, stored, orotherwise, combined at any step in the methods of the procedure with agrowth medium comprising one or more inhibitors a component of a PI3Kpathway. Exemplary inhibitors a component of a PI3K pathway include, butare not limited to, an inhibitor of GSK3β such as TWS119 (also known asGSK 3B inhibitor XII; CAS Number 601514-19-6 having a chemical formulaC₁₈H₁₄N₄O₂). Exemplary inhibitors a component of a PI3K pathway include,but are not limited to, bb007 (BLUEBIRDBIO™).

As used herein, the terms “electroporation” and “nucleofection” aremeant to describe alternative means to deliver a nucleic acid,transposon, vector or composition of the disclosure to a cell byproviding an electric pulse that induces a cell membrane (the cellmembrane, nuclear membrane, or both) to become permeable or to becomemore permeable to the nucleic acid, transposon, vector or composition ofthe disclosure.

In certain embodiments of the nucleofection, the method is performed oneor more cuvette(s) simultaneously. In certain embodiments of thenucleofection, the method is performed in two cuvettes simultaneously.For a process performed on a larger scale for clinical or commercialapplications, for example, the nucleofections may be performed in alarge-volume cassette with many procedures ongoing simultaneously. Incertain embodiments of the nucleofection, the incubating step comprisesincubating the composition comprising the plurality of primary human Tcells in a pre-warmed T-cell expansion composition. The incubation stepmay have a period of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 hours, or any number/portionof hours in between. The incubation step may have a period of at least1, 2, 3, 4, 5, 6 or 7 days or any number/portion of days in between. Theincubation step may have a period of at least 1 week. In certainembodiments of the nucleofection, the incubation step has a period oftwo days. In certain embodiments of the nucleofection, the applying stepmay comprise applying one or more of the following program(s) EI-115,EI-151, EI-156, EI-158, EG-115, EG-142, EG-151, ES-115, ES-151, EO-151,EO-148, EO-156, EO-210, EO-213, and FI-156. In certain embodiments, theapplying step may comprise applying one or more of the followingprogram(s) EI-115, EI-151, EI-156, EI-158, EG-115, EG-142, EG-151,ES-115, ES-151, EO-151, EO-148, EO-156, EO-210, EO-213, and FI-156, or aprogram that provides the same number of electrical pulses, each pulsehaving the same duration and intensity, and a substantially similarinterpulse duration of time. In certain embodiments, the applying stepmay be performed using a known electroporation/nucleofection device,including, but not limited to, Lonza Amaxa, MaxCyte technology, BTXPulseAgile, and BioRad GenePulser. In certain embodiments of thenucleofection, the applying step may comprise applying at least oneelectrical pulse. In certain embodiments of the nucleofection, theapplying step may comprise applying at least one electrical pulsesufficient to induce the cell membrane and/or nuclear membrane of a cellto become permeable to a composition of the disclosure.

While the amounts provided herein are exemplary and non-limiting, therelationship between these amounts (e.g. ratios or relative abundances)may be used to modify the methods exemplified herein for larger-scaleprocesses and manufacturing.

In certain embodiments of the methods of producing a modified T cell(e.g. a T_(SCM) and/or T_(CM)) of the disclosure, the activationsupplement comprises one or more cytokine(s). The one or morecytokine(s) may comprise any cytokine, including but not limited to,lymphokines. Exemplary lympokines include, but are not limited to,interleukin-2 (IL-2), interleukin-3 (IL-3), interleukin-4 (IL-4),interleukin-5 (IL-5), interleukin-6 (IL-6), interleukin-7 (IL-7),interleukin-15 (IL-15), interleukin-21 (IL-21), granulocyte-macrophagecolony-stimulating factor (GM-CSF) and interferon-gamma (INFy). The oneor more cytokine(s) may comprise IL-2.

In certain embodiments of the methods of producing a modified T cell(e.g. a T_(SCM) and/or T_(CM)) of the disclosure, the expansionsupplement comprises one or more cytokine(s). The one or morecytokine(s) may comprise any cytokine, including but not limited to,lymphokines. Exemplary lympokines include, but are not limited to,interleukin-2 (IL-2), interleukin-3 (IL-3), interleukin-4 (IL-4),interleukin-5 (IL-5), interleukin-6 (IL-6), interleukin-7 (IL-7),interleukin-15 (IL-15), interleukin-21 (IL-21), granulocyte-macrophagecolony-stimulating factor (GM-CSF) and interferon-gamma (INFy). The oneor more cytokine(s) may comprise IL-2.

In certain embodiments of the methods of producing a modified T cell(e.g. a T_(SCM) and/or T_(CM)) of the disclosure, the primary human Tcell is a naïve T cell. The naïve T cell may express CD45RA, CCR7 andCD62L. In certain embodiments, the method is applied to a cellpopulation comprising at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%,45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or anypercentage in between of naïve T cells. In certain embodiments, theefficiency of production of modified T_(SCM) and/or T_(CM) of thedisclosure may be increased by increasing a proportion or percentage ofnaïve T cells in a cell population to which the methods of thedisclosure are applied.

In certain embodiments of the methods of producing a modified T_(SCM)and/or T_(CM) of the disclosure, the primary human T cell is a memory Tcell.

In certain embodiments of the methods of producing a modified T_(SCM)and/or T_(CM) of the disclosure, the primary human T cell expresses oneor more of CD62L, CD45RA, CD28, CCR7, CD127, CD45RO, CD95, CD95 andIL-2Rβ.

In certain embodiments of the methods of producing a modified T_(SCM)and/or T_(CM) of the disclosure, the primary human T cell is a naïveT-cell (modified T_(N)) and the modified T_(N) expresses one or more ofCD45RA, CCR7 and CD62L. In certain embodiments of the methods ofproducing a modified T_(SCM) and/or T_(CM) of the disclosure, theprimary human T cell is a modified T_(SCM) a T memory stem cell(modified T_(SCM)) and the modified T_(SCM) expresses one or more ofCD45RA, CD95, IL-2Rβ, CR7, and CD62L. In certain embodiments of themethods of producing a modified T_(SCM) and/or T_(CM) of the disclosure,the primary human T cell is a central memory T-cell (modified T_(CM))and the modified T_(CM) expresses one or more of CD45RO, CD95, IL-2Rβ,CCR7, and CD62L. In certain embodiments of the methods of producing amodified T_(SCM) and/or T_(CM) of the disclosure, the primary human Tcell is an effector memory T-cell (modified T_(EM)) and the modifiedT_(EM) expresses one or more of CD45RO, CD95, and IL-2Rβ. In certainembodiments of the methods of producing a modified T_(SCM) and/or T_(CM)of the disclosure, the primary human T cell is an effector T-cell(modified T_(EFF)) and the modified T_(EFF) expresses one or more ofCD45RA, CD95, and IL-2Rβ.

In certain embodiments of the methods of producing a modified T_(SCM)and/or T_(CM) of the disclosure, the primary human T cell may expressCD4 and/or CD8. In certain embodiments, the primary human T cell mayexpress CD4 and/or CD8 at various ratios. In certain embodiments, theprimary human T cell may express CD4 and/or CD8 at various ratios thatare not naturally-occurring. In certain embodiments, the primary human Tcells that express CD4 and/or CD8 at various ratios, that may be notnaturally occurring, are a heterologous cell population.

In certain embodiments of the methods of producing a modified T_(SCM)and/or T_(CM) of the disclosure, the primary human T cell may beisolated, prepared or derived from for example, whole blood, peripheralblood, umbilical cord blood, lymph fluid, lymph node tissue, bonemarrow, and cerebral spinal fluid (CSF). The term “peripheral blood” asused herein, refers to cellular components of blood (e.g., red bloodcells, white blood cells and platelets), which are obtained or preparedfrom the circulating pool of blood and not sequestered within thelymphatic system, spleen, liver or bone marrow. Umbilical cord blood isdistinct from peripheral blood and blood sequestered within thelymphatic system, spleen, liver or bone marrow. The terms “umbilicalcord blood”, “umbilical blood” or “cord blood”, which can be usedinterchangeably, refers to blood that remains in the placenta and in theattached umbilical cord after child birth. Cord blood often containsstem cells including hematopoietic cells.

Primary human T cells of the disclosure may comprise pan T cells. Asused herein, pan T-cells include all T lymphocytes isolated from abiological sample, without sorting by subtype, activation status,maturation state, or cell-surface marker expression.

In certain embodiments of the methods of the disclosure, the methodfurther comprises introducing into a modified T_(SCM) or T_(CM) cell acomposition comprising a genomic editing construct or composition. Incertain embodiments, the genomic editing construct comprises a guide RNAand a clustered regularly interspaced short palindromic repeats (CRISPR)associated protein 9 (Cas9) DNA endonuclease. In certain embodiments,the genomic editing construct comprises a DNA binding domain and a typeIIS endonuclease. In certain embodiments, the genomic editing constructencodes a fusion protein. In certain embodiments, the genomic editingconstruct encodes the DNA binding domain and the type IIS endonucleaseand wherein the expressed DNA binding domain and the expressed type IISendonuclease are non-covalently linked. In certain embodiments,including those embodiments wherein the genomic editing constructcomprises a DNA binding domain and a type IIS endonuclease, the genomicediting construct comprises a sequence derived from a Cas9 endonuclease.In certain embodiments, including those embodiments wherein the genomicediting construct comprises a DNA binding domain and a type IISendonuclease, the sequence derived from a Cas9 endonuclease is the DNAbinding domain. In certain embodiments, including those embodimentswherein the sequence derived from a Cas9 endonuclease is the DNA bindingdomain, the sequence derived from a Cas9 endonuclease encodes aninactive Cas9. In certain embodiments, including those embodimentswherein the sequence derived from a Cas9 endonuclease is the DNA bindingdomain, the sequence derived from a Cas9 endonuclease encodes atruncated Cas9. In certain embodiments, the sequence derived from a Cas9endonuclease comprises an amino acid substitution of an Alanine (A) foran Aspartic Acid (D) at position 10 (D10A). In certain embodiments, thesequence derived from a Cas9 endonuclease comprises or further comprisesan amino acid substitution of an Alanine (A) for a Histidine (H) atposition 840 (H840A). In certain embodiments, the sequence derived froma Cas9 endonuclease comprises an inactivated Cas9 (dCas9) (SEQ ID NO:33). In certain embodiments, the sequence derived from a Cas9endonuclease comprises an amino acid substitution of an alanine (A) foran Asparagine (N) at position 580 (N580A). In certain embodiments, thesequence derived from a Cas9 endonuclease comprises a truncated andinactivated Cas9 (dSaCas9) (SEQ ID NO: 32). In certain embodiments,including those embodiments wherein the genomic editing constructcomprises a DNA binding domain and a type IIS endonuclease, the genomicediting construct comprises a sequence derived from a transcriptionactivator-like effector nuclease (TALEN). In certain embodiments,including those embodiments wherein the genomic editing constructcomprises a DNA binding domain and a type IIS endonuclease, the sequencederived from a TALEN is the DNA binding domain. In certain embodiments,the genomic editing construct comprises a TALEN. In certain embodiments,including those embodiments wherein the genomic editing constructcomprises a DNA binding domain and a type IIS endonuclease, the genomicediting construct comprises a sequence derived from a zinc-fingernuclease (ZFN). In certain embodiments, including those embodimentswherein the genomic editing construct comprises a DNA binding domain anda type IIS endonuclease, the sequence derived from a ZFN is the DNAbinding domain. In certain embodiments, the genomic editing constructcomprises a zinc-finger nuclease (ZFN).

The methods of making modified T_(SCM) and/or T_(CM) cells of thedisclosure may be optimized to produce a greater number or greaterproportion of modified T_(SCM) and/or T_(CM) cells. For example, thepopulation of cells subjected to the methods of the disclosure may beenriched to contain an increased number or greater proportion of naïve Tcells. As the number and/or proportion of naïve T cells increases in thepopulation of T cells subjected to the methods of the disclosure, thenumber and/or proportion of modified T_(SCM) and/or T_(CM) cells of thedisclosure produced also increases. Alternatively, or in addition, asthe length of time or duration required for a method of disclosure toprecede decreases, the number and/or proportion of modified T_(SCM)and/or T_(CM) cells of the disclosure produced by the method increases.The length of time or duration required for a method of disclosure toprecede, or the “manufacturing period” may also be referred to as the“out-of-life period” of the T cells subjected to the methods of thedisclosure.

In certain embodiments of the methods of making modified T-cells of thedisclosure, the primary human T cell expresses one or more of CD62L,CD45RA, CD28, CCR7, CD127, CD45RO, CD95, CD95 and IL-2Rβ. In certainembodiments, the primary human T cell is a naïve T-cell (T_(N)) and theT_(N) expresses one or more of CD45RA, CCR7 and CD62L. In certainembodiments, the primary human T cell is a T memory stem cell (T_(SCM))and the T_(SCM) expresses one or more of CD45RA, CD95, IL-2Rβ, CR7, andCD62L. In certain embodiments, the primary human T cell is a centralmemory T-cell (T_(CM)) and wherein the T_(CM) expresses one or more ofCD45RO, CD95, IL-2Rβ, CCR7, and CD62L. In certain embodiments, theprimary human T cell is an effector memory T-cell (T_(EM)) and the EMexpresses one or more of CD45RO, CD95, and IL-2Rβ. In certainembodiments, the primary human T cell is an effector T-cell (T_(EFF))and the T_(EFF) expresses one or more of CD45RA, CD95, and IL-2Rβ. Incertain embodiments, the primary human T cell expresses CD4 and/or CD8.

The disclosure provides a composition comprising a modified T_(SCM)produced a method of the disclosure. The disclosure provides acomposition comprising a modified T_(CM) produced a method of thedisclosure. The disclosure provides a composition comprising a modifiedT_(SCM) and a modified T_(CM) produced a method of the disclosure. Incertain embodiments of the composition comprising a modified T_(SCM) anda modified T_(CM) produced a method of the disclosure, a plurality ofT_(SCM) may comprise at least 1%, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%,40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or99% or the composition. In certain embodiments of the compositioncomprising a modified T_(SCM) and a modified T_(CM) produced a method ofthe disclosure, a plurality of T_(CM) may comprise at least 1%, 2%, 5%,10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, 95%, 97%, 98% or 99% or the composition.

The disclosure provides a use of a composition comprising a modifiedT_(SCM) and/or T_(CM) produced a method of the disclosure for themanufacture of a medicament to treat a subject in need thereof. Incertain embodiments of this use, the modified T_(SCM) and/or T_(CM) isautologous. In certain embodiments of this use, the modified T_(SCM)and/or T_(CM) is allogeneic. In certain embodiments, the antigenreceptor is a T-cell receptor. In certain embodiments, the T-cellreceptor is naturally-occurring. In certain embodiments, the T-cellreceptor is not naturally-occurring. In certain embodiments, and, inparticular, in those embodiments wherein the T-cell receptor is notnaturally-occurring, the T-cell receptor comprises one or moremutation(s) compared to a wild-type T-cell receptor. In certainembodiments, and, in particular, in those embodiments wherein the T-cellreceptor is not naturally-occurring, the T-cell receptor is arecombinant T-cell receptor. In certain embodiments, the antigenreceptor is a Chimeric Antigen Receptor (CAR). In certain embodiments,the CAR is a CARTyrin. In certain embodiments, the CAR comprises one ormore VHH sequence(s). In certain embodiments, the CAR is a VCAR.

The disclosure provides a method of treating a disease or disorder in asubject in need thereof, comprising administering to the subject atherapeutically effective amount of a composition comprising a modifiedT_(SCM) and/or T_(CM) produced a method of the disclosure. In certainembodiments of this method, the modified T_(SCM) and/or T_(CM) isautologous. In certain embodiments of this method, the modified T_(SCM)and/or T_(CM) is allogeneic. In certain embodiments, the antigenreceptor is a T-cell receptor. In certain embodiments, the T-cellreceptor is naturally-occurring. In certain embodiments, the T-cellreceptor is not naturally-occurring. In certain embodiments, and, inparticular, in those embodiments wherein the T-cell receptor is notnaturally-occurring, the T-cell receptor comprises one or moremutation(s) compared to a wild-type T-cell receptor. In certainembodiments, and, in particular, in those embodiments wherein the T-cellreceptor is not naturally-occurring, the T-cell receptor is arecombinant T-cell receptor. In certain embodiments, the antigenreceptor is a Chimeric Antigen Receptor (CAR). In certain embodiments,the CAR is a CARTyrin. In certain embodiments, the CAR comprises one ormore VHH sequence(s). In certain embodiments, the CAR is a VCAR. Incertain embodiments of this method, the disease or disorder is cancerand the antigen receptor specifically targets a cancer antigen. Incertain embodiments of this method, the disease or disorder is aninfectious disease or disorder and the antigen receptor specificallytargets a viral, bacterial, yeast or microbial antigen. In certainembodiments, the disease or disorder is a disease or disorder caused bya lack of an activity or an insufficient amount of a secretory protein.In certain embodiments, the disease or disorder is a disease or disordertreated by a replacement of an activity of a therapeutic protein or byan increase in an amount of the therapeutic protein. In certainembodiments, the therapeutic protein is a secreted protein. In certainembodiments, the secretory protein is lacking an activity or asufficient amount within a local area of a body. In certain embodiments,the local area of a body is accessible by a native T-cell or a modifiedT-cell. In certain embodiments, the modified T-cell is produced in vivo,ex vivo, in vitro or in situ.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a series of plots depicting the emergence of the CAR-T_(SCM)phenotype at Day 11 of the method of Example 1. Cells were nucleofectedwith a surrogate CARTyrin plasmid. CAR-T_(SCM) cells express CD62L andCD45RA as shown in the bottom two plots.

FIG. 2 is a series of plots depicting the purity of the CAR-T_(SCM)produced by the method of Example 1 at day 19. The population ofCAR-T_(SCM) cells produced by the method described in Example 1 at day19 contained no B cells or lymphocytes. The majority of the cells areCD3+ T-cells. Only 1.1% are Natural Killer cells and 1.7% are NaturalKiller T-cells.

FIG. 3 is a plot showing that at Day 11 of the method described inExample 1, the majority of the T-cells produced express the CARTyrin.

FIG. 4 is a series of plots depicting an enrichment of the CAR-T_(SCM)phenotype at Day 19 of the method described in Example 1. Cells werenucleofected with a surrogate CARTyrin plasmid. CAR-T_(SCM) cellsexpress CD62L and CD45RA as shown in the bottom two plots.

FIG. 5 is a series of plots depicting the absence of T-cell exhaustionat Day 19 of the method described in Example 1. At Day 19, the cellpopulation produced by this method does not express PD1, which is amarker for T cell activation and exhaustion. These cells expressing theCARTyrin have almost successfully reached a resting statepost-manufacture. They do not exhibit signs of antigen-independent(tonic) signaling which would otherwise drive higher levels of PD1expression. Tonic signaling is hypothesized to be caused by some CARmolecules that lead to early exhaustion and reduced efficacy of a CART-cell therapy.

FIG. 6A is a series of plots depicting T cells transposed with a plasmidcontaining a sequence encoding a transposon comprising a sequenceencoding an inducible caspase polypeptide (a safety switch, “iC9”), aCARTyrin (anti-BCMA), and a selectable marker. Left-hand plots depictlive T cells exposed to transposase in the absence of the plasmid.Right-hand plots depict live T cells exposed to transposase in thepresence of the plasmid. Cells were exposed to either a hyperactivetransposase (the “Super piggyBac”) or a wild type piggyBac transposase.

FIG. 6B is a series of plots depicting T cells transposed with a plasmidcontaining a sequence encoding a green fluorescent protein (GFP).Left-hand plots depict live T cells exposed to transposase in theabsence of the plasmid. Right-hand plots depict live T cells exposed totransposase in the presence of the plasmid. Cells were exposed to eithera hyperactive transposase (the “Super piggyBac”) or a wild type piggyBactransposase.

FIG. 6C is a table depicting the percent of transformed T cellsresulting from transposition with WT versus hyperactive piggyBactransposase. T cells contacted with the hyperactive piggyBac transposase(the Super piggyBac transposase) were transformed at a rate 4-foldgreater than WT transposase.

FIG. 6D is a graph depicting the percent of transformed T cellsresulting from transposition with WT versus hyperactive piggyBactransposase 5 days after nucleofection. T cells contacted with thehyperactive piggyBac transposase (the Super piggyBac transposase) weretransformed at a rate far greater than WT transposase.

FIG. 7 is a graph showing a phenotypic difference between piggyBac™- andlentivirus-produced CAR+ T cells. CAR+ T cells were produced usingeither piggyBac transposition or lentivirus transduction. Human pan Tcells were transposed with piggyBac encoding CAR, stimulated withanti-CD3/CD28 beads at day 2 post-transposition, expanded, and examinedon day 19 post-transposition. For production using lentivirus, pan Tcells were stimulated with aCD3/CD28 beads, transduced with lentivirusencoding CAR (MOI 5), expanded, and examined on day 18 post-stimulation.Then, each population of CAR+ T cells was characterized based on theirexpression of the standard memory markers CD62L, CD45RA and CD95. Thepercentage of each CAR+ T cell subset was defined as naïve(CD62L+CD45RA+), Tcm (CD62L+CD45RA−), Tem (CD62L-CD45RA−) and Teff(CD62L-CD45RA+). All CAR+ T cells were CD95+.

FIG. 8A-B is a pair of graphs showing that piggyBac™ preferentiallytransposes naïve T cells. Human pan T cells were sorted (using a BDFACSAria II flow cytometer) into naïve (CD62L+CD45RA+), Tcm(CD62L+CD45RA−), Tem (CD62L-CD45RA−), and Teff (CD62L-CD45RA+) subsets.The sorted subsets were each either transposed with piggyBac-GFP ortransduced with lentivirus-GFP. For the former, each sorted subset wastransposed with PiggyBac-GFP, stimulated with anti-CD3/CD28 beads at day2 post-transposition, expanded, and examined on day 19post-transposition. For the latter, the sorted subsets were stimulatedwith aCD3/CD28 beads, transduced with lentivirus encoding GFP (MOI 5),expanded, and examined on day 19 post-stimulation. n=3 donors.

FIG. 9 is a pair of graphs showing that the piggyBac™ manufacturingprocess yields high levels of T_(SCM) in samples from multiple myeloma(MM) patients even when naïve T cells are rare. T cells from MM patients(triangles) and healthy donors (circles) were characterized for memorymarker expression by flow cytometry before (left) and after (right) thePoseida manufacturing process. Expression of CD45RA and CD62L wasassessed by FACS and plots are shown for the MM patients and a healthydonor. It is known that T cells from MM patients generally have lowerfrequencies of naïve and T_(SCM) cells, but higher frequencies of Teff,unlike those from healthy normal donors which are the opposite.Regardless of the input frequency of naïve and T_(SCM) from different MMpatients, production of P-BCMA-101 using the Poseida manufacturingprocess resulted in a product that exhibited a high level of CD8+ Tscm(E). This was also true for a MM patient who was actively receivingtreatment (red triangle).

FIG. 10A-B is a series of Fluorescence Activated Cell Sorting (FACs)plots characterizing T and T_(SCM) cell markers in human pan T cellstransformed with the Sleeping Beauty (SB100x) transposition system andthe methods of the disclosure. Sleeping Beauty (SB100x) Transpositionyields predominately T_(SCM) phenotype using Poseida manufactureprocess. Human pan T cells were transposed using 1 μg of either aSleeping Beauty or piggyBac transposon plasmid and SB100x or SPB mRNA,respectively as shown. Following transposition, cells were expanded exvivo and all non-transposed cells were depleted using the Poseidamanufacture drug selection system. Following 18 days in culture, cellswere stained with the phenotypic markers CD4, CD8, CD45RA, and CD62L.Stem cell memory phenotype (T_(SCM)) is defined by CD45RA and CD62Ldouble positive cells and make up >65% of the cells in all of samples.All panels in a column share common x-axis and y-axis parameters. Ineach row, from top to bottom, are shown data from T cells transposedwith (top), 2.5 microgram (μg) of the Sleeping Beauty transposon SB100x,(second from top) 5 μg of SB100x, (3^(rd) from top) 10 μg of SB100x,(second from bottom) 5 μg of the piggyBac transposon P-BCMA-101 and atbottom, an unstained control. The x-axis, in order from left to right,in the first and second columns shows Forward Scatter (FSC), units from0 to 250 thousand (abbreviated “k”), in increments of 50 k. The x axisof the third column from the left shows CD8 expression, with markingsreading from 0 to 10⁵ incrementing by powers of 10. The final right handcolumn shows CD62L expression, with markings reading from 0 to 10⁵incrementing by powers of 10. The y-axis, in the first column, showsSide Scatter (SSC), in units from 0 to 250 k in increments of 50 k. They-axis in the second column from the left shows expression of the cellviability marker 7 aminoactinomycin D (7AAD), from 0 to 10⁵ incrementingby powers of 10. The y-axis of the third column from the left shows theexpression of the marker CD4, from 0 to 10⁵ incrementing by powers of10. The y-axis in the right hand column show expression of the markerCD45RA, from 0 to 10⁵ incrementing by powers of 10.

FIG. 11 is a schematic diagram showing the human coagulation pathwayleading to blood clotting. Contact activation, for example by damagingan endothelium, activates an intrinsic clotting pathway. Tissue factorsactivate an extrinsic clotting pathway, for example following trauma.Both pathways converge onto the conversion of Prothrombin into Thrombin,which catalyzes the conversion of fibrinogen into fibrin. Polymerizedfibrin together with platelets forms a clot. In the absence of Factor IX(circled), clotting is defective. Factor VIII (FVIII) deficiency leadsto development of Hemophilia A. Factor IX (FIX) deficiency leads todevelopment of Hemophilia B. Hemophilia B is a rare disease, occurringwith a frequency of about one in between 25,000 and 30,000. Sixtypercent of hemophilia B cases are severe. Fewer than one percent ofindividuals with Hemophilia B have normal FIX levels. Prior to thecompositions and methods of the disclosure, the standard treatment forhemophilia B involved an infusion of recombinant FIX every 2 to 3 days,at an expense of approximately $250,000 per year. In sharp contrast tothis standard treatment option, T_(SCM) cells of the disclosure aremaintained in humans for several decades.

FIG. 12 is a series of Fluorescence-Activated Cell Sorting (FACS plots)depicting FIX-secreting T cells. T cells encoding a human Factor IXtransgene showed a T_(SCM) phenotype in approximately 80% of cells. The6 panels are described in order from left to right. (1) Forward scatter(FSC) on the x-axis versus side scatter (SSC) on the y-axis. The x-axisis from 0 to 250 thousand (abbreviated k) in increments of 50 k, they-axis is for 0 to 250 k, in increments of 50 k. (2) FSC on the x-axisversus the cell viability marker 7 aminoactinomycin D (7AAD). The x-axisis labeled from 0 to 250 k in increments of 50 k. The y-axis reads, fromtop to bottom, −10³, 0, 10³, 10⁴, 10⁵. (3) On the x-axis is shownanti-CD56-APC conjugated to a Cy7 dye (CDC56-APC-Cy7), units from 0 to10⁵ incrementing in powers of 10. On the y-axis is shown anti-CD3conjugated to phycoerythrin (PE), units from 0 to 10⁵ incrementing inpowers of 10. (4) On the x-axis is shown anti-CD8 conjugated tofluorescein isothiocyanate (FITC), units from 0 to 10⁵ incrementing inpowers of 10. On the y-axis is shown anti-CD4 conjugated to BrilliantViolet 650 dye (BV650), units from 0 to 10⁵ incrementing in powers of10. (5) On the x-axis is shown an anti CD62L antibody conjugated to aBrilliant Violet 421 dye (BV421), units from 0 to 10⁵ incrementing inpowers of 10. On the y-axis is shown an anti-CD45RA antibody conjugatedto PE and Cy7, units from 0 to 10⁵ incrementing in powers of 10. Thispanel is boxed. (6) On the x-axis is shown an anti-CCR7 antibodyconjugated to Brilliant Violet 786 (BV786), units from 0 to 10⁵incrementing in powers of 10. On the y-axis is shown anti-CD45RAconjugated to PE and Cy7, units from 0 to 10⁵ incrementing in powers of10.

FIG. 13A is a graph showing human Factor IX secretion during productionof modified T cells of the disclosure. On the y-axis, Factor IXconcentration in nanograms (ng) per milliliter (mL) from 0 to 80 inincrements of 20. On the x-axis are shown 9 day and 12 day T cells.

FIG. 13B is a graph showing the clotting activity of the secreted FactorIX produced by the T cells. On the y-axis is shown percent Factor IXactivity relative to human plasma, from 0 to 8 in increments of 2. Onthe x-axis are 9 and 12 day T cells.

FIGS. 14A-E are a series of plasmid maps for site-specific integrationinto the AAVS1 site using either HR or MMEJ and corresponding sequences.Donor plasmids for testing stable integration into the genome of humanpan T cells via A) site-specific (AAVS1) homologous recombination (HR),B) site-specific (AAVS1) microhomology-mediated end-joining (MMEJ)recombination and C) TTAA-specific piggyBac™ transposition. For HR andMMEJ donor plasmids, GFP-2A-DHFR gene expression cassettes were flankedby CRISPR/Cas9 targeting sites and homology arms for AAVS1 siteintegration; for piggyBac™ donor plasmid, GFP-2A-DHFR gene expressioncassette is flanked by piggyBac™ transposon elements. The homology armsfor the HR and MMEJ plasmids are 500 bp and 25 bp, respectively. PanelsD and E depict SEQ ID NOs 41 and 42 respectively.

FIG. 15 is a graph showing transgene (GFP) expression in primary humanpan T cells 3 days post-nucleofection. HR or MMEJ donor plasmids wereco-delivered with or without CRISPR ribonucleoprotein (RNP) targetingreagents into pan T cells via nucleofection. T cells receiving donorplasmids alone were included as controls. Pan T cells were also modifiedusing the piggyBac™ transposon delivery system. T cells were activatedvia TCR stimulation on Day 0 and GFP+ T cell percentage was accessed atday 3 post-nucleofection by flow cytometry and data are summarized inbar graph.

FIG. 16 is a graph showing transgene (GFP) expression in primary humanpan T cells 11 days post-nucleofection and selection. Activated T cellswith stably integrated transgenes were selected by methotrexate additionusing the DHFR selection gene encoded in the bi-cistronic GFP-2A-DHFRintegration cassettes. GFP+ cell percentage was assessed at Day 11post-nucleofection by flow cytometry and data are summarized in bargraph. GFP+ cells were highly enriched via selection in pan T cellsreceiving transposition reagents, RNP plus HR or MMEJ donor plasmids,but not in T cells receiving donor plasmids alone.

FIG. 17A-C is a series of graphs showing the phenotype of primary humanpan T cells modified by HR and MMEJ at the AAVS1 site. The phenotype ofGFP+CD8+ pan T cells was analyzed at Day 11 post-nucleofection by flowcytometry. A) Cells were stained with 7AAD (cell viability), CD4, CD8,CD45RA and CD62L, and FACS plots show gating strategy. CD8+ T cellsubsets were defined by expression of CD45RA+CD62L+(stem cell memory Tcells (T_(SCM))), CD45RA-CD62L+(central memory T cells (T_(CM))),CD45RA-CD62L-(effector memory T cells (Tem)), and CD45RA+CD62L-(Teffectors (Teff)). B) Percentage of total GFP+CD8+ T cells in each Tcell subset is summarized in bar graph. An enriched population of GFP+Tscm was achieved in all cases using either the piggyBac™ transposonsystem, or HR and MMEJ in combination with Cas9 RNP. C) The total numberof pan T cells was analyzed at day 13 post-nucleofection and data aresummarized in bar graph.

FIG. 18A-B is a pair of photographs of gel electrophoresis resultsshowing site-specific integration into the AAVS1 site. Selected cellsfrom each group were harvested and genomic DNA was extracted and used astemplate for PCR to confirm site-specific integration into the AAVS1site for A) HR and B) MMEJ. Two pairs of primers individually amplifythe 5′-end junction (with one primer priming the promoter region of theinsertion EFla-2r CACCGGAGCCAATTCCCACT (SEQ ID NO: 36) and the otherpriming the AAVS1 region beyond the 500 bp homologue arm at the 5′-endAAVS-3r CTGCACCACGTGATGTCCTC (SEQ ID NO: 37), yielding a 0.73 kb DNAfragment for both HR or MMEJ) and 3′-end junction (with one primerpriming the polyA signaling region SV40 pA-1rGTAACCATTATAAGCTGCAATAAACAAG (SEQ ID NO: 38) and the other priming theAAVS1 region beyond the 500 bp 5′-homologue arm AAVS-2fCTGGGGACTCTTTAAGGAAAGAAG (SEQ ID NO: 39), yielding a 0.76 kb DNAfragment for HR or MMEJ) of the AAVS1 target site. PCR products weredisplayed on Agarose gel. Non-specific bands in HR samples are theresult of only a single round of PCR and would likely have been resolvedgiven additional rounds.

DETAILED DESCRIPTION

The disclosure provides a method for producing human chimeric antigenreceptor (CAR) expressing-T cells using the piggyBac™ Transposon Systemunder conditions that preserve or induce stem-cell memory T cells(T_(SCM)) with potent CAR activity (referred to herein as a CAR-T_(SCM).Compositions comprising CAR-T_(SCM) produced using the methods of thedisclosure comprise ≥60% CAR-T_(SCM) and exhibit a distinct functionalprofile that is consistent with this T cell subset. Other T cell subsetsfound in the compositions of the disclosure include, but are not limitedto, central memory CAR-T cells (CAR-T_(CM)), effector memory CAR-T cells(CAR-T_(EM)), effector CAR-T cells (CAR-T_(E)), andterminally-differentiated effector CAR-T cells (CAR-TTE). A linearpathway of differentiation may be responsible for generating thesecells: Naïve T cells (T_(N))>T_(SCM)>T_(CM)>T_(EM)>T_(E)>T_(TE), wherebyT_(N) is the parent precursor cell that directly gives rise to T_(SCM),which then, in turn, directly gives rise to T_(CM), etc. Compositionscomprising CAR-T_(SCM), CARTyrin-T_(SCM) and/or VCAR-T_(SCM) of thedisclosure may comprise one or more of each parental CAR-T cell subsetwith CAR-T_(SCM) being the most abundant (e.g.T_(SCM)>T_(CM)>T_(EM)>T_(E)>T_(TE)). While, the absolutequantities/abundances and relative proportions of each parental T cellsubset may vary among samples of patient blood and naturally-occurringcell populations, and naturally-occurring cell populations may have ahigh abundance and/or proportion of T_(SCM), compositions of thedisclosure comprising non-naturally occurring CAR-T_(SCM) are morepotent and efficacious in treating patients against diseases andcancers.

Immunotherapy using chimeric-antigen receptor (CAR)-T cells is emergingas an exciting therapeutic approach for cancer therapies. AutologousCAR-modified T cells targeting a tumor-associated antigen (Ag) canresult in robust tumor killing, in some cases resulting in completeremission of CD19⁺ hematological malignancies. Unlike traditionalbiologics and chemotherapeutics, CAR-T cells possess the capacity torapidly reproduce upon Ag recognition, thereby potentially obviating theneed for repeat treatments. To achieve this, CAR-T cells must not onlydrive tumor destruction initially, but must also persist in the patientas a stable population of viable memory T cells to prevent potentialcancer relapses. Thus, intensive efforts have been focused on thedevelopment of CAR molecules that do not cause T cell exhaustion throughAg-independent (tonic) signaling, as well as of a CAR-T productcontaining early memory cells, especially stem cell memory (T_(SCM)). Astem cell-like CAR-T would exhibit the greatest capacity forself-renewal and multipotent capacity to derive central memory (T_(CM)),effector memory (T_(EM)) and effector T cells (T_(E)), thereby producingbetter tumor eradication and long-term CAR-T engraftment.

CAR-T_(SCM) of the disclosure may comprise a Centyrin-based CAR,referred to as a CARTyrin (and hence, the cell may be referred to as aCARTyrin-T_(SCM)). Centyrins are alternative scaffold molecules based onhuman consensus tenascin FN3 domain, are smaller than scFv molecules,and can be selected for monomeric properties that favor stability anddecrease the likelihood of tonic signaling in CAR molecules. CARTyrinsof the disclosure may be introduced to T cells using a plasmid DNAtransposon encoding the CARTyrin that is flanked by two cis-regulatoryinsulator elements to help stabilize CARTyrin expression by blockingimproper gene activation or silencing.

CAR-T_(SCM) of the disclosure may comprise a VHH-based CAR, referred toas a VCAR (and hence, the cell may be referred to as a VCAR-T_(SCM))VCARs of the disclosure may be introduced to T cells using a plasmid DNAtransposon encoding the VHH that is flanked by two cis-regulatoryinsulator elements to help stabilize VHH expression by blocking impropergene activation or silencing.

In certain embodiments of the methods of the disclosure, the piggyBac™(PB) Transposon System may be used for stable integration ofantigen-specific (including cancer antigen-specific) CARTyrin or VCARinto resting pan T cells, whereby the transposon was co-delivered alongwith an mRNA transposase enzyme (although the transposon and transposasewould be comprised in separate compositions until they were introducedinto a cell), called Super piggyBac™ (SPB), in a single electroporationreaction. Delivery of piggyBac™ transposon into untouched, restingprimary human pan T cells resulted in 20-30% of cells with stableintegration and expression of PB-delivered genes. Unexpectedly, amajority of these modified CARTyrin-expressing T cells were positive forexpression of CD62L and CD45RA, markers commonly associated with stemmemory T-cells (T_(SCM) cells). To confirm that this phenotype wasretained upon CAR-T cell stimulation and expansion, the modifiedCARTyrin-expressing T cells positive for expression of CD62L and CD45RAwere activated via stimulation of CD3 and CD28. As a result ofstimulation of CD3 and CD28, >60% of CARTyrin+ T cells exhibited astem-cell memory phenotype. Furthermore, these cells, which expressed aCARTyrin specific for a cancer antigen, were fully capable of expressingpotent anti-tumor effector function.

To determine whether or not the PB system directly contributed toenhancing the expression of stem-like markers, the phenotype of CAR-Tcells generated either by PB transposition or lentiviral (LV)transduction was compared. To do this, a new vector was constructed bysubcloning the CARTyrin transgene into a common LV construct forproduction of virus. Following introduction of the CARTyrin to untouchedresting T cells either by PB-transposition or LV-transduction, theCARTyrin⁺ cells were expanded and then allowed to return to a restingstate. A variety of phenotypic and functional characteristics weremeasured including kinetic analysis of memory and exhaustion-associatedmarkers, secondary proliferation in response to homeostatic cytokine ortumor-associated Ag, cytokine production, and lytic capability inresponse to target tumor cells. Unlike the PB-transposed CARTyrin⁺ Tcells, the LV-transduced CARTyrin⁺ T cells did not exhibit an augmentedmemory phenotype. In addition, PB-transposed cells exhibited acomparable or greater capability for secondary proliferation and killingof target tumor cells. Together, these data demonstrate that CAR-T cellsproduced by PB transposition are predominantly T_(SCM) cells, a highlydesirable product phenotype in the CAR-T field. Furthermore, theseCARTyrin T cells exhibit strong anti-tumor activity and may give rise tocells that persist longer in vivo due to the use of a Centyrin-basedCAR, which may be less prone to tonic signaling and functionalexhaustion.

Chimeric Antigen Receptors

The disclosure provides a chimeric antigen receptor (CAR) comprising:(a) an ectodomain comprising an antigen recognition region, wherein theantigen recognition region comprises one or more sequences that eachspecifically bind an antigen; (b) a transmembrane domain, and (c) anendodomain comprising at least one costimulatory domain. In certainembodiments, the antigen recognition region may comprise two sequencesthat each specifically bind an antigen to produce a bi-specific ortandem CAR. In certain embodiments, the antigen recognition region maycomprise three sequences that each specifically bind an antigen toproduce a tri-specific CAR. In certain embodiments, the ectodomain mayfurther comprise a signal peptide. Alternatively, or in addition, incertain embodiments, the ectodomain may further comprise a hinge betweenthe antigen recognition region and the transmembrane domain. Sequencesthat each specifically bind an antigen may include, but not limited to,a single chain antibody (e.g. a scFv), a sequence comprising one or morefragments of an antibody (e.g. a VHH, referred to in the context of aCAR as a VCAR), an antibody mimic, and a Centyrin (referred to in thecontext of a CAR as a CARTyrin).

In certain embodiments of the CARs of the disclosure, the signal peptidemay comprise a sequence encoding a human CD2, CD3δ, CD3ε, CD3γ, CD3ζ,CD4, CD8α, CD19, CD28, 4-1BB or GM-CSFR signal peptide. In certainembodiments of the CARs of the disclosure, the signal peptide maycomprise a sequence encoding a human CD8a signal peptide. The human CD8asignal peptide may comprise an amino acid sequence comprisingMALPVTALLLPLALLLHAARP (SEQ ID NO: 8). The human CD8a signal peptide maycomprise an amino acid sequence comprising MALPVTALLLPLALLLHAARP (SEQ IDNO: 8) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identityto the an amino acid sequence comprising MALPVTALLLPLALLLHAARP (SEQ IDNO: 8). The human CD8a signal peptide may be encoded by a nucleic acidsequence comprisingatggcactgccagtcaccgccctgctgctgcctctggctctgctgctgcacgcagctagacca (SEQ IDNO: 9).

In certain embodiments of the CARs of the disclosure, the transmembranedomain may comprise a sequence encoding a human CD2, CD3δ, CD3ε, CD3γ,CD3ζ, CD4, CD8α, CD19, CD28, 4-1BB or GM-CSFR transmembrane domain. Incertain embodiments of the CARs of the disclosure, the transmembranedomain may comprise a sequence encoding a human CD8α transmembranedomain. The CD8α transmembrane domain may comprise an amino acidsequence comprising IYIWAPLAGTCGVLLLSLVITLYC (SEQ ID NO: 10) or asequence having at least 70%, 80%, 90%, 95%, or 99% identity to theamino acid sequence comprising IYIWAPLAGTCGVLLLSLVITLYC (SEQ ID NO: 10).The CD8a transmembrane domain may be encoded by the nucleic acidsequence comprisingatctacatttgggcaccactggccgggacctgtggagtgctgctgctgagcctggtcatcacactgtactgc(SEQ ID NO: 11).

In certain embodiments of the CARs of the disclosure, the endodomain maycomprise a human CD3ζ endodomain.

In certain embodiments of the CARs of the disclosure, the at least onecostimulatory domain may comprise a human 4-1BB, CD28, CD40, ICOS,MyD88, OX-40 intracellular segment, or any combination thereof. Incertain embodiments of the CARs of the disclosure, the at least onecostimulatory domain may comprise a CD28 and/or a 4-1BB costimulatorydomain. The CD28 costimulatory domain may comprise an amino acidsequence comprisingRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 12)or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to theamino acid sequence comprisingRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGL STATKDTYDALHMQALPPR (SEQ ID NO: 12).The CD28 costimulatory domain may be encoded by the nucleic acidsequence comprisingcgcgtgaagtttagtcgatcagcagatgccccagcttacaaacagggacagaaccagctgtataacgagctgaatctgggccgccgagaggaatatgacgtgctggataagcggagaggacgcgaccccgaaatgggaggcaagcccaggcgcaaaaaccctcaggaaggcctgtataacgagctgcagaaggacaaaatggcagaagcctattctgagatcggcatgaagggggagcgacggagaggcaaagggcacgatgggctgtaccagggactgagcaccgccacaaaggacacctatgatgctctgcatatgcaggcactgcctccaagg(SEQ ID NO: 13). The 4-1BB costimulatory domain may comprise an aminoacid sequence comprising KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQID NO: 14) or a sequence having at least 70%, 80%, 90%, 95%, or 99%identity to the amino acid sequence comprisingKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO: 14). The 4-1BBcostimulatory domain may be encoded by the nucleic acid sequencecomprisingaagagaggcaggaagaaactgctgtatattttcaaacagccatcatgcgccccgtgcagactacccaggaggaagacgggtgctcctgtcgattccctgaggaagaggaaggcgggtgtgagctg (SEQ ID NO: 15). The 4-1BBcostimulatory domain may be located between the transmembrane domain andthe CD28 costimulatory domain.

In certain embodiments of the CARs of the disclosure, the hinge maycomprise a sequence derived from a human CD8α, IgG4, and/or CD4sequence. In certain embodiments of the CARs of the disclosure, thehinge may comprise a sequence derived from a human CD8a sequence. Thehinge may comprise a human CD8a amino acid sequence comprisingTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO: 16) or asequence having at least 70%, 80%, 90%, 95%, or 99% identity to theamino acid sequence comprisingTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO: 16). The humanCD8a hinge amino acid sequence may be encoded by the nucleic acidsequence comprisingactaccacaccagcacctagaccaccaactccagctccaaccatcgcgagtcagcccctgagtctgagacctgaggcctgcaggccagctgcaggaggagctgtgcacaccaggggcctggacttcgcctgcgac (SEQ ID NO: 17).

The disclosure provides a composition comprising the CAR of thedisclosure and at least one pharmaceutically acceptable carrier.

The disclosure provides a transposon comprising the CAR of thedisclosure. Transposons of the disclosure be episomally maintained orintegrated into the genome of the recombinant/modified cell. Thetransposon may be part of a two component piggyBac system that utilizesa transposon and transposase for enhanced non-viral gene transfer.

Transposons of the disclosure may comprise a selection gene foridentification, enrichment and/or isolation of cells that express thetransposon. Exemplary selection genes encode any gene product (e.g.transcript, protein, enzyme) essential for cell viability and survival.Exemplary selection genes encode any gene product (e.g. transcript,protein, enzyme) essential for conferring resistance to a drug challengeagainst which the cell is sensitive (or which could be lethal to thecell) in the absence of the gene product encoded by the selection gene.Exemplary selection genes encode any gene product (e.g. transcript,protein, enzyme) essential for viability and/or survival in a cell medialacking one or more nutrients essential for cell viability and/orsurvival in the absence of the selection gene. Exemplary selection genesinclude, but are not limited to, neo (conferring resistance toneomycin), DHFR (encoding Dihydrofolate Reductase and conferringresistance to Methotrexate), TYMS (encoding Thymidylate Synthetase),MGMT (encoding O(6)-methylguanine-DNA methyltransferase), multidrugresistance gene (MDR1), ALDH1 (encoding Aldehyde dehydrogenase 1 family,member A1), FRANCF, RAD51C (encoding RAD51 Paralog C), GCS (encodingglucosylceramide synthase), and NKX2.2 (encoding NK2 Homeobox 2).

Transposons of the disclosure may comprise at least one self-cleavingpeptide(s) located, for example, between one or more of a sequence thatspecifically binds an antigen and a selection gene of the disclosure.The at least one self-cleaving peptide may comprise, for example, a T2Apeptide, GSG-T2A peptide, an E2A peptide, a GSG-E2A peptide, an F2Apeptide, a GSG-F2A peptide, a P2A peptide, or a GSG-P2A peptide. A T2Apeptide may comprise an amino acid sequence comprisingEGRGSLLTCGDVEENPGP (SEQ ID NO: 18) or a sequence having at least 70%,80%, 90%, 95%, or 99% identity to the amino acid sequence comprisingEGRGSLLTCGDVEENPGP (SEQ ID NO: 18). A GSG-T2A peptide may comprise anamino acid sequence comprising GSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 19) ora sequence having at least 70%, 80%, 90%, 95%, or 99% identity to theamino acid sequence comprising GSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 19). AGSG-T2A peptide may comprise a nucleic acid sequence comprisingggatctggagagggaaggggaagcctgctgacctgtggagacgtggaggaaaacccaggacca (SEQ IDNO: 20). An E2A peptide may comprise an amino acid sequence comprisingQCTNYALLKLAGDVESNPGP (SEQ ID NO: 21) or a sequence having at least 70%,80%, 90%, 95%, or 99% identity to the amino acid sequence comprisingQCTNYALLKLAGDVESNPGP (SEQ ID NO: 21). A GSG-E2A peptide may comprise anamino acid sequence comprising GSGQCTNYALLKLAGDVESNPGP (SEQ ID NO: 22)or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to theamino acid sequence comprising GSGQCTNYALLKLAGDVESNPGP (SEQ ID NO: 22).An F2A peptide may comprise an amino acid sequence comprisingVKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 23) or a sequence having at least70%, 80%, 90%, 95%, or 99% identity to the amino acid sequencecomprising VKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 23). A GSG-F2A peptide maycomprise an amino acid sequence comprising GSGVKQTLNFDLLKLAGDVESNPGP(SEQ ID NO: 24) or a sequence having at least 70%, 80%, 90%, 95%, or 99%identity to the amino acid sequence comprising GSGVKQTLNFDLLKLAGDVESNPGP(SEQ ID NO: 24). A P2A peptide may comprise an amino acid sequencecomprising ATNFSLLKQAGDVEENPGP (SEQ ID NO: 25) or a sequence having atleast 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequencecomprising ATNFSLLKQAGDVEENPGP (SEQ ID NO: 25). A GSG-P2A peptide maycomprise an amino acid sequence comprising GSGATNFSLLKQAGDVEENPGP (SEQID NO: 26) or a sequence having at least 70%, 80%, 90%, 95%, or 99%identity to the amino acid sequence comprising GSGATNFSLLKQAGDVEENPGP(SEQ ID NO: 26).

Transposons of the disclosure may comprise a first and a secondself-cleaving peptide, the first self-cleaving peptide located, forexample, upstream of one or more of a sequence that specifically bindsan antigen of the disclosure the second self-cleaving peptide located,for example, downstream of the one or more of a sequence thatspecifically binds an antigen of the disclosure. The first and/or thesecond self-cleaving peptide may comprise, for example, a T2A peptide,GSG-T2A peptide, an E2A peptide, a GSG-E2A peptide, an F2A peptide, aGSG-F2A peptide, a P2A peptide, or a GSG-P2A peptide. A T2A peptide maycomprise an amino acid sequence comprising EGRGSLLTCGDVEENPGP (SEQ IDNO: 18) or a sequence having at least 70%, 80%, 90%, 95%, or 99%identity to the amino acid sequence comprising EGRGSLLTCGDVEENPGP (SEQID NO: 18). A GSG-T2A peptide may comprise an amino acid sequencecomprising GSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 19) or a sequence having atleast 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequencecomprising GSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 19). A GSG-T2A peptide maycomprise a nucleic acid sequence comprisingggatctggagagggaaggggaagcctgctgacctgtggagacgtggaggaaaacccaggacca (SEQ IDNO: 20). An E2A peptide may comprise an amino acid sequence comprisingQCTNYALLKLAGDVESNPGP (SEQ ID NO: 21) or a sequence having at least 70%,80%, 90%, 95%, or 99% identity to the amino acid sequence comprisingQCTNYALLKLAGDVESNPGP (SEQ ID NO: 21). A GSG-E2A peptide may comprise anamino acid sequence comprising GSGQCTNYALLKLAGDVESNPGP (SEQ ID NO: 22)or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to theamino acid sequence comprising GSGQCTNYALLKLAGDVESNPGP (SEQ ID NO: 22).An F2A peptide may comprise an amino acid sequence comprisingVKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 23) or a sequence having at least70%, 80%, 90%, 95%, or 99% identity to the amino acid sequencecomprising VKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 023). A GSG-F2A peptidemay comprise an amino acid sequence comprising GSGVKQTLNFDLLKLAGDVESNPGP(SEQ ID NO: 24) or a sequence having at least 70%, 80%, 90%, 95%, or 99%identity to the amino acid sequence comprising GSGVKQTLNFDLLKLAGDVESNPGP(SEQ ID NO: 24). A P2A peptide may comprise an amino acid sequencecomprising ATNFSLLKQAGDVEENPGP (SEQ ID NO: 25) or a sequence having atleast 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequencecomprising ATNFSLLKQAGDVEENPGP (SEQ ID NO: 25). A GSG-P2A peptide maycomprise an amino acid sequence comprising GSGATNFSLLKQAGDVEENPGP (SEQID NO: 26) or a sequence having at least 70%, 80%, 90%, 95%, or 99%identity to the amino acid sequence comprising GSGATNFSLLKQAGDVEENPGP(SEQ ID NO: 26).

The disclosure provides a composition comprising the transposon thedisclosure. In certain embodiments, a method introducing the compositionmay further comprise a composition comprising a plasmid comprising asequence encoding a transposase enzyme. The sequence encoding atransposase enzyme may be an mRNA sequence.

Transposons of the disclosure may comprise piggyBac transposons.Transposase enzymes of the disclosure may include piggyBac transposasesor compatible enzymes.

The disclosure provides a vector comprising the CAR of the disclosure.In certain embodiments, the vector is a viral vector. The vector may bea recombinant vector.

Viral vectors of the disclosure may comprise a sequence isolated orderived from a retrovirus, a lentivirus, an adenovirus, anadeno-associated virus or any combination thereof. The viral vector maycomprise a sequence isolated or derived from an adeno-associated virus(AAV). The viral vector may comprise a recombinant AAV (rAAV). Exemplaryadeno-associated viruses and recombinant adeno-associated viruses of thedisclosure comprise two or more inverted terminal repeat (ITR) sequenceslocated in cis next to one or more of a sequence that specifically bindsan antigen. Exemplary adeno-associated viruses and recombinantadeno-associated viruses of the disclosure include, but are not limitedto all serotypes (e.g. AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8,and AAV9). Exemplary adeno-associated viruses and recombinantadeno-associated viruses of the disclosure include, but are not limitedto, self-complementary AAV (scAAV) and AAV hybrids containing the genomeof one serotype and the capsid of another serotype (e.g. AAV2/5, AAV-DJand AAV-DJ8). Exemplary adeno-associated viruses and recombinantadeno-associated viruses of the disclosure include, but are not limitedto, rAAV-LK03.

Viral vectors of the disclosure may comprise a selection gene. Theselection gene may encode a gene product essential for cell viabilityand survival. The selection gene may encode a gene product essential forcell viability and survival when challenged by selective cell cultureconditions. Selective cell culture conditions may comprise a compoundharmful to cell viability or survival and wherein the gene productconfers resistance to the compound. Exemplary selection genes of thedisclosure may include, but are not limited to, neo (conferringresistance to neomycin), DHFR (encoding Dihydrofolate Reductase andconferring resistance to Methotrexate), TYMS (encoding ThymidylateSynthetase), MGMT (encoding O(6)-methylguanine-DNA methyltransferase),multidrug resistance gene (MDR1), ALDH1 (encoding Aldehyde dehydrogenase1 family, member A1), FRANCF, RAD51C (encoding RAD51 Paralog C), GCS(encoding glucosylceramide synthase), NKX2.2 (encoding NK2 Homeobox 2)or any combination thereof.

Viral vectors of the disclosure may comprise at least one self-cleavingpeptide. In some embodiments, the vector may comprise at least oneself-cleaving peptide and wherein a self-cleaving peptide is locatedbetween a CAR and a selection gene. In some embodiments, the vector maycomprise at least one self-cleaving peptide and wherein a firstself-cleaving peptide is located upstream of a CAR and a secondself-cleaving peptide is located downstream of a CAR. The self-cleavingpeptide may comprise, for example, a T2A peptide, GSG-T2A peptide, anE2A peptide, a GSG-E2A peptide, an F2A peptide, a GSG-F2A peptide, a P2Apeptide, or a GSG-P2A peptide. A T2A peptide may comprise an amino acidsequence comprising EGRGSLLTCGDVEENPGP (SEQ ID NO: 18) or a sequencehaving at least 70%, 80%, 90%, 95%, or 99% identity to the amino acidsequence comprising EGRGSLLTCGDVEENPGP (SEQ ID NO: 18). A GSG-T2Apeptide may comprise an amino acid sequence comprisingGSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 19) or a sequence having at least 70%,80%, 90%, 95%, or 99% identity to the amino acid sequence comprisingGSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 19). A GSG-T2A peptide may comprise anucleic acid sequence comprisingggatctggagagggaaggggaagcctgctgacctgtggagacgtggaggaaaacccaggacca (SEQ IDNO: 20). An E2A peptide may comprise an amino acid sequence comprisingQCTNYALLKLAGDVESNPGP (SEQ ID NO: 21) or a sequence having at least 70%,80%, 90%, 95%, or 99% identity to the amino acid sequence comprisingQCTNYALLKLAGDVESNPGP (SEQ ID NO: 21). A GSG-E2A peptide may comprise anamino acid sequence comprising GSGQCTNYALLKLAGDVESNPGP (SEQ ID NO: 22)or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to theamino acid sequence comprising GSGQCTNYALLKLAGDVESNPGP (SEQ ID NO: 22).An F2A peptide may comprise an amino acid sequence comprisingVKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 23) or a sequence having at least70%, 80%, 90%, 95%, or 99% identity to the amino acid sequencecomprising VKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 23). A GSG-F2A peptide maycomprise an amino acid sequence comprising GSGVKQTLNFDLLKLAGDVESNPGP(SEQ ID NO: 24) or a sequence having at least 70%, 80%, 90%, 95%, or 99%identity to the amino acid sequence comprising GSGVKQTLNFDLLKLAGDVESNPGP(SEQ ID NO: 24). A P2A peptide may comprise an amino acid sequencecomprising ATNFSLLKQAGDVEENPGP (SEQ ID NO: 25) or a sequence having atleast 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequencecomprising ATNFSLLKQAGDVEENPGP (SEQ ID NO: 25). A GSG-P2A peptide maycomprise an amino acid sequence comprising GSGATNFSLLKQAGDVEENPGP (SEQID NO: 26) or a sequence having at least 70%, 80%, 90%, 95%, or 99%identity to the amino acid sequence comprising GSGATNFSLLKQAGDVEENPGP(SEQ ID NO: 26).

The disclosure provides a vector comprising the CAR of the disclosure.In certain embodiments, the vector is an mRNA vector. The vector may bea recombinant mRNA vector. T cells of the disclosure may be expandedprior to contacting the T-cell and the mRNA vector comprising the CAR ofthe disclosure. The T cell comprising the mRNA vector, the modified Tcell, may then be administered to a subject.

The disclosure provides a vector comprising the CAR of the disclosure.In certain embodiments, the vector is a nanoparticle. Exemplarynanoparticle vectors of the disclosure include, but are not limited to,nucleic acids (e.g. RNA, DNA, synthetic nucleotides, modifiednucleotides or any combination thereof), amino acids (L-amino acids,D-amino acids, synthetic amino acids, modified amino acids, or anycombination thereof), polymers (e.g. polymersomes), micelles, lipids(e.g. liposomes), organic molecules (e.g. carbon atoms, sheets, fibers,tubes), inorganic molecules (e.g. calcium phosphate or gold) or anycombination thereof. A nanoparticle vector may be passively or activelytransported across a cell membrane.

Nanoparticle vectors of the disclosure may comprise a selection gene.The selection gene may encode a gene product essential for cellviability and survival. The selection gene may encode a gene productessential for cell viability and survival when challenged by selectivecell culture conditions. Selective cell culture conditions may comprisea compound harmful to cell viability or survival and wherein the geneproduct confers resistance to the compound. Exemplary selection genes ofthe disclosure may include, but are not limited to, neo (conferringresistance to neomycin), DHFR (encoding Dihydrofolate Reductase andconferring resistance to Methotrexate), TYMS (encoding ThymidylateSynthetase), MGMT (encoding O(6)-methylguanine-DNA methyltransferase),multidrug resistance gene (MDR1), ALDH1 (encoding Aldehyde dehydrogenase1 family, member A1), FRANCF, RAD51C (encoding RAD51 Paralog C), GCS(encoding glucosylceramide synthase), NKX2.2 (encoding NK2 Homeobox 2)or any combination thereof.

Nanoparticle vectors of the disclosure may comprise at least oneself-cleaving peptide. In some embodiments, the nanoparticle vector maycomprise at least one self-cleaving peptide and wherein a self-cleavingpeptide is located between a CAR and the nanoparticle. In someembodiments, the nanoparticle vector may comprise at least oneself-cleaving peptide and wherein a first self-cleaving peptide islocated upstream of a CAR and a second self-cleaving peptide is locateddownstream of a CAR. In some embodiments, the nanoparticle vector maycomprise at least one self-cleaving peptide and wherein a firstself-cleaving peptide is located between a CAR and the nanoparticle anda second self-cleaving peptide is located downstream of the CAR. In someembodiments, the nanoparticle vector may comprise at least oneself-cleaving peptide and wherein a first self-cleaving peptide islocated between a CAR and the nanoparticle and a second self-cleavingpeptide is located downstream of the CAR, for example, between the CARand a selection gene. The self-cleaving peptide may comprise, forexample, a T2A peptide, GSG-T2A peptide, an E2A peptide, a GSG-E2Apeptide, an F2A peptide, a GSG-F2A peptide, a P2A peptide, or a GSG-P2Apeptide. A T2A peptide may comprise an amino acid sequence comprisingEGRGSLLTCGDVEENPGP (SEQ ID NO: 18) or a sequence having at least 70%,80%, 90%, 95%, or 99% identity to the amino acid sequence comprisingEGRGSLLTCGDVEENPGP (SEQ ID NO: 18). A GSG-T2A peptide may comprise anamino acid sequence comprising GSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 19) ora sequence having at least 70%, 80%, 90%, 95%, or 99% identity to theamino acid sequence comprising GSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 19). AGSG-T2A peptide may comprise a nucleic acid sequence comprisingggatctggagagggaaggggaagcctgctgacctgtggagacgtggaggaaaacccaggacca (SEQ IDNO: 20). An E2A peptide may comprise an amino acid sequence comprisingQCTNYALLKLAGDVESNPGP (SEQ ID NO: 21) or a sequence having at least 70%,80%, 90%, 95%, or 99% identity to the amino acid sequence comprisingQCTNYALLKLAGDVESNPGP (SEQ ID NO: 21). A GSG-E2A peptide may comprise anamino acid sequence comprising GSGQCTNYALLKLAGDVESNPGP (SEQ ID NO: 22)or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to theamino acid sequence comprising GSGQCTNYALLKLAGDVESNPGP (SEQ ID NO: 22).An F2A peptide may comprise an amino acid sequence comprisingVKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 23) or a sequence having at least70%, 80%, 90%, 95%, or 99% identity to the amino acid sequencecomprising VKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 23). A GSG-F2A peptide maycomprise an amino acid sequence comprising GSGVKQTLNFDLLKLAGDVESNPGP(SEQ ID NO: 24) or a sequence having at least 70%, 80%, 90%, 95%, or 99%identity to the amino acid sequence comprising GSGVKQTLNFDLLKLAGDVESNPGP(SEQ ID NO: 24). A P2A peptide may comprise an amino acid sequencecomprising ATNFSLLKQAGDVEENPGP (SEQ ID NO: 25) or a sequence having atleast 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequencecomprising ATNFSLLKQAGDVEENPGP (SEQ ID NO: 25). A GSG-P2A peptide maycomprise an amino acid sequence comprising GSGATNFSLLKQAGDVEENPGP (SEQID NO: 26) or a sequence having at least 70%, 80%, 90%, 95%, or 99%identity to the amino acid sequence comprising GSGATNFSLLKQAGDVEENPGP(SEQ ID NO: 26).

The disclosure provides a composition comprising a vector of thedisclosure.

CARTyrins

The disclosure provides a chimeric antigen receptor (CAR) comprising:(a) an ectodomain comprising an antigen recognition region, wherein theantigen recognition region comprises at least one Centyrin; (b) atransmembrane domain, and (c) an endodomain comprising at least onecostimulatory domain. As used throughout the disclosure, a CARcomprising a Centyrin is referred to as a CARTyrin. In certainembodiments, the antigen recognition region may comprise two Centyrinsto produce a bi-specific or tandem CAR. In certain embodiments, theantigen recognition region may comprise three Centyrins to produce atri-specific CAR. In certain embodiments, the ectodomain may furthercomprise a signal peptide. Alternatively, or in addition, in certainembodiments, the ectodomain may further comprise a hinge between theantigen recognition region and the transmembrane domain.

The disclosure provides a chimeric antigen receptor (CAR) comprising:(a) an ectodomain comprising an antigen recognition region, wherein theantigen recognition region comprises at least one protein scaffold orantibody mimetic; (b) a transmembrane domain, and (c) an endodomaincomprising at least one costimulatory domain. In certain embodiments,the antigen recognition region may comprise two scaffold proteins orantibody mimetics to produce a bi-specific or tandem CAR. In certainembodiments, the antigen recognition region may comprise three proteinscaffolds or antibody mimetics to produce a tri-specific CAR. In certainembodiments, the ectodomain may further comprise a signal peptide.Alternatively, or in addition, in certain embodiments, the ectodomainmay further comprise a hinge between the antigen recognition region andthe transmembrane domain.

In certain embodiments of the CARs of the disclosure, the signal peptidemay comprise a sequence encoding a human CD2, CD3δ, CD3ε, CD3γ, CD3ζ,CD4, CD8α, CD19, CD28, 4-1BB or GM-CSFR signal peptide. In certainembodiments of the CARs of the disclosure, the signal peptide maycomprise a sequence encoding a human CD8a signal peptide. The human CD8asignal peptide may comprise an amino acid sequence comprisingMALPVTALLLPLALLLHAARP (SEQ ID NO: 8). The human CD8a signal peptide maycomprise an amino acid sequence comprising MALPVTALLLPLALLLHAARP (SEQ IDNO: 8) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identityto the an amino acid sequence comprising MALPVTALLLPLALLLHAARP (SEQ IDNO: 8). The human CD8a signal peptide may be encoded by a nucleic acidsequence comprisingatggcactgccagtcaccgccctgctgctgcctctggctctgctgctgcacgcagctagacca (SEQ IDNO: 9).

In certain embodiments of the CARs of the disclosure, the transmembranedomain may comprise a sequence encoding a human CD2, CD3δ, CD3ε, CD3γ,CD3ζ, CD4, CD8α, CD19, CD28, 4-1BB or GM-CSFR transmembrane domain. Incertain embodiments of the CARs of the disclosure, the transmembranedomain may comprise a sequence encoding a human CD8a transmembranedomain. The CD8α transmembrane domain may comprise an amino acidsequence comprising IYIWAPLAGTCGVLLLSLVITLYC (SEQ ID NO: 10) or asequence having at least 70%, 80%, 90%, 95%, or 99% identity to theamino acid sequence comprising IYIWAPLAGTCGVLLLSLVITLYC (SEQ ID NO: 10).The CD8α transmembrane domain may be encoded by the nucleic acidsequence comprisingatctacatttgggcaccactggccgggacctgtggagtgctgctgctgagcctggtcatcacactgtactgc(SEQ ID NO: 11).

In certain embodiments of the CARs of the disclosure, the endodomain maycomprise a human CD3ζ endodomain.

In certain embodiments of the CARs of the disclosure, the at least onecostimulatory domain may comprise a human 4-1BB, CD28, CD40, ICOS,MyD88, OX-40 intracellular segment, or any combination thereof. Incertain embodiments of the CARs of the disclosure, the at least onecostimulatory domain may comprise a CD28 and/or a 4-1BB costimulatorydomain. The CD28 costimulatory domain may comprise an amino acidsequence comprisingRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGL STATKDTYDALHMQALPPR (SEQ ID NO: 12)or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to theamino acid sequence comprisingRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGL STATKDTYDALHMQALPPR (SEQ ID NO: 12).The CD28 costimulatory domain may be encoded by the nucleic acidsequence comprisingcgcgtgaagtttagtcgatcagcagatgccccagcttacaaacagggacagaaccagctgtataacgagctgaatctgggccgccgagaggaatatgacgtgctggataagcggagaggacgcgaccccgaaatgggaggcaagcccaggcgcaaaaaccctcaggaaggcctgtataacgagctgcagaaggacaaaatggcagaagcctattctgagatcggcatgaagggggagcgacggagaggcaaagggcacgatgggctgtaccagggactgagcaccgccacaaaggacacctatgatgctctgcatatgcaggcactgcctccaagg(SEQ ID NO: 13). The 4-1BB costimulatory domain may comprise an aminoacid sequence comprising KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQID NO: 14) or a sequence having at least 70%, 80%, 90%, 95%, or 99%identity to the amino acid sequence comprisingKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO: 14). The 4-1BBcostimulatory domain may be encoded by the nucleic acid sequencecomprisingaagagaggcaggaagaaactgctgtatattttcaaacagccatcatgcgccccgtgcagactacccaggaggaagacgggtgctcctgtcgattccctgaggaagaggaaggcgggtgtgagctg (SEQ ID NO: 15). The 4-1BBcostimulatory domain may be located between the transmembrane domain andthe CD28 costimulatory domain.

In certain embodiments of the CARs of the disclosure, the hinge maycomprise a sequence derived from a human CD8α, IgG4, and/or CD4sequence. In certain embodiments of the CARs of the disclosure, thehinge may comprise a sequence derived from a human CD8α sequence. Thehinge may comprise a human CD8α amino acid sequence comprisingTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO: 16) or asequence having at least 70%, 80%, 90%, 95%, or 99% identity to theamino acid sequence comprisingTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO: 16). The humanCD8α hinge amino acid sequence may be encoded by the nucleic acidsequence comprisingactaccacaccagcacctagaccaccaactccagctccaaccatcgcgagtcagcccctgagtctgagacctgaggcctgcaggccagctgcaggaggagctgtgcacaccaggggcctggacttcgcctgcgac (SEQ ID NO: 17).

Centyrins of the disclosure may comprise a protein scaffold, wherein thescaffold is capable of specifically binding an antigen. Centyrins of thedisclosure may comprise a protein scaffold comprising a consensussequence of at least one fibronectin type III (FN3) domain, wherein thescaffold is capable of specifically binding an antigen. The at least onefibronectin type III (FN3) domain may be derived from a human protein.The human protein may be Tenascin-C. The consensus sequence may compriseLPAPKNLVVSEVTEDSLRLSWTAPDAAFDSFLIQYQESEKVGEAINLTVPGSERSYDLTGLKPGTEYTVSIYGVKGGHRSNPLSAEFTT (SEQ ID NO: 1) orMLPAPKNLVVSEVTEDSLRLSWTAPDAAFDSFLIQYQESEKVGEAINLTVPGSERSYDLTGLKPGTEYTVSIYGVKGGHRSNPLSAEFTT (SEQ ID NO: 2). The consensus sequencemay encoded by a nucleic acid sequence comprisingatgctgcctgcaccaaagaacctggtggtgtctcatgtgacagaggatagtgccagactgtcatggactgctcccgacgcagccttcgatagttttatcatcgtgtaccgggagaacatcgaaaccggcgaggccattgtcctgacagtgccagggtccgaacgctatatgacctgacagatctgaagcccggaactgagtactatgtgcagatcgccggcgtcaaaggaggcaatatcagatccctctgtccgcaatcttcaccaca(SEQ ID NO: 3). The consensus sequence may be modified at one or morepositions within (a) a A-B loop comprising or consisting of the aminoacid residues TEDS (SEQ ID NO: 43) at positions 13-16 of the consensussequence; (b) a B-C loop comprising or consisting of the amino acidresidues TAPDAAF (SEQ ID NO: 44) at positions 22-28 of the consensussequence; (c) a C-D loop comprising or consisting of the amino acidresidues SEKVGE (SEQ ID NO: 45) at positions 38-43 of the consensussequence; (d) a D-E loop comprising or consisting of the amino acidresidues GSER (SEQ ID NO: 46) at positions 51-54 of the consensussequence; (e) a E-F loop comprising or consisting of the amino acidresidues GLKPG (SEQ ID NO: 47) at positions 60-64 of the consensussequence; (f) a F-G loop comprising or consisting of the amino acidresidues KGGHRSN (SEQ ID NO: 48) at positions 75-81 of the consensussequence; or (g) any combination of (a)-(f). Centyrins of the disclosuremay comprise a consensus sequence of at least 5 fibronectin type III(FN3) domains, at least 10 fibronectin type III (FN3) domains or atleast 15 fibronectin type III (FN3) domains. The scaffold may bind anantigen with at least one affinity selected from a K_(D) of less than orequal to 10⁻⁹M, less than or equal to 10⁻¹⁰ M, less than or equal to10⁻¹¹ M, less than or equal to 10⁻¹² M, less than or equal to 10⁻¹³ M,less than or equal to 10⁻¹⁴ M, and less than or equal to 10⁻¹⁵ M. TheK_(D) may be determined by surface plasmon resonance.

The disclosure provides a composition comprising the CAR of thedisclosure and at least one pharmaceutically acceptable carrier.

The disclosure provides a transposon comprising the CAR of thedisclosure.

Transposons of the disclosure be episomally maintained or integratedinto the genome of the recombinant/modified cell. The transposon may bepart of a two component piggyBac system that utilizes a transposon andtransposase for enhanced non-viral gene transfer.

Transposons of the disclosure may comprise a selection gene foridentification, enrichment and/or isolation of cells that express thetransposon. Exemplary selection genes encode any gene product (e.g.transcript, protein, enzyme) essential for cell viability and survival.Exemplary selection genes encode any gene product (e.g. transcript,protein, enzyme) essential for conferring resistance to a drug challengeagainst which the cell is sensitive (or which could be lethal to thecell) in the absence of the gene product encoded by the selection gene.Exemplary selection genes encode any gene product (e.g. transcript,protein, enzyme) essential for viability and/or survival in a cell medialacking one or more nutrients essential for cell viability and/orsurvival in the absence of the selection gene. Exemplary selection genesinclude, but are not limited to, neo (conferring resistance toneomycin), DHFR (encoding Dihydrofolate Reductase and conferringresistance to Methotrexate), TYMS (encoding Thymidylate Synthetase),MGMT (encoding O(6)-methylguanine-DNA methyltransferase), multidrugresistance gene (MDR1), ALDH1 (encoding Aldehyde dehydrogenase 1 family,member A1), FRANCF, RAD51C (encoding RAD51 Paralog C), GCS (encodingglucosylceramide synthase), and NKX2.2 (encoding NK2 Homeobox 2).

Transposons of the disclosure may comprise at least one self-cleavingpeptide(s) located, for example, between on or more of a proteinscaffold, Centyrin or CARTyrin of the disclosure and a selection gene ofthe disclosure. The at least one self-cleaving peptide may comprise, forexample, a T2A peptide, GSG-T2A peptide, an E2A peptide, a GSG-E2Apeptide, an F2A peptide, a GSG-F2A peptide, a P2A peptide, or a GSG-P2Apeptide. A T2A peptide may comprise an amino acid sequence comprisingEGRGSLLTCGDVEENPGP (SEQ ID NO: 18) or a sequence having at least 70%,80%, 90%, 95%, or 99% identity to the amino acid sequence comprisingEGRGSLLTCGDVEENPGP (SEQ ID NO: 18). A GSG-T2A peptide may comprise anamino acid sequence comprising GSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 19) ora sequence having at least 70%, 80%, 90%, 95%, or 99% identity to theamino acid sequence comprising GSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 19). AGSG-T2A peptide may comprise a nucleic acid sequence comprisingggatctggagagggaaggggaagcctgctgacctgtggagacgtggaggaaaacccaggacca (SEQ IDNO: 20). An E2A peptide may comprise an amino acid sequence comprisingQCTNYALLKLAGDVESNPGP (SEQ ID NO: 21) or a sequence having at least 70%,80%, 90%, 95%, or 99% identity to the amino acid sequence comprisingQCTNYALLKLAGDVESNPGP (SEQ ID NO: 21). A GSG-E2A peptide may comprise anamino acid sequence comprising GSGQCTNYALLKLAGDVESNPGP (SEQ ID NO: 22)or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to theamino acid sequence comprising GSGQCTNYALLKLAGDVESNPGP (SEQ ID NO: 22).An F2A peptide may comprise an amino acid sequence comprisingVKQTLNFDLLKLAGDVESNPGP (SEQ NO: 23) or a sequence having at least 70%,80%, 90%, 95%, or 99% identity to the amino acid sequence comprisingVKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 23). A GSG-F2A peptide may comprisean amino acid sequence comprising GSGVKQTLNFDLLKLAGDVESNPGP (SEQ ID NO:24) or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity tothe amino acid sequence comprising GSGVKQTLNFDLLKLAGDVESNPGP (SEQ ID NO:24). A P2A peptide may comprise an amino acid sequence comprisingATNFSLLKQAGDVEENPGP (SEQ ID NO: 25) or a sequence having at least 70%,80%, 90%, 95%, or 99% identity to the amino acid sequence comprisingATNFSLLKQAGDVEENPGP (SEQ ID NO: 25). A GSG-P2A peptide may comprise anamino acid sequence comprising GSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 26) ora sequence having at least 70%, 80%, 90%, 95%, or 99% identity to theamino acid sequence comprising GSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 26).

Transposons of the disclosure may comprise a first and a secondself-cleaving peptide, the first self-cleaving peptide located, forexample, upstream of one or more of a protein scaffold, Centyrin orCARTyrin of the disclosure the second self-cleaving peptide located, forexample, downstream of the one or more of a protein scaffold, Centyrinor CARTyrin of the disclosure. The first and/or the second self-cleavingpeptide may comprise, for example, a T2A peptide, GSG-T2A peptide, anE2A peptide, a GSG-E2A peptide, an F2A peptide, a GSG-F2A peptide, a P2Apeptide, or a GSG-P2A peptide. A T2A peptide may comprise an amino acidsequence comprising EGRGSLLTCGDVEENPGP (SEQ ID NO: 18) or a sequencehaving at least 70%, 80%, 90%, 95%, or 99% identity to the amino acidsequence comprising EGRGSLLTCGDVEENPGP (SEQ ID NO: 18). A GSG-T2Apeptide may comprise an amino acid sequence comprisingGSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 19) or a sequence having at least 70%,80%, 90%, 95%, or 99% identity to the amino acid sequence comprisingGSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 19). A GSG-T2A peptide may comprise anucleic acid sequence comprisingggatctggagagggaaggggaagcctgctgacctgtggagacgtggaggaaaacccaggacca (SEQ IDNO: 20). An E2A peptide may comprise an amino acid sequence comprisingQCTNYALLKLAGDVESNPGP (SEQ ID NO: 21) or a sequence having at least 70%,80%, 90%, 95%, or 99% identity to the amino acid sequence comprisingQCTNYALLKLAGDVESNPGP (SEQ ID NO:21). A GSG-E2A peptide may comprise anamino acid sequence comprising GSGQCTNYALLKLAGDVESNPGP (SEQ ID NO: 22)or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to theamino acid sequence comprising GSGQCTNYALLKLAGDVESNPGP (SEQ ID NO: 22).An F2A peptide may comprise an amino acid sequence comprisingVKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 23) or a sequence having at least70%, 80%, 90%, 95%, or 99% identity to the amino acid sequencecomprising VKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 23). A GSG-F2A peptide maycomprise an amino acid sequence comprising GSGVKQTLNFDLLKLAGDVESNPGP(SEQ ID NO: 24) or a sequence having at least 70%, 80%, 90%, 95%, or 99%identity to the amino acid sequence comprising GSGVKQTLNFDLLKLAGDVESNPGP(SEQ ID NO: 24). A P2A peptide may comprise an amino acid sequencecomprising ATNFSLLKQAGDVEENPGP (SEQ ID NO: 25) or a sequence having atleast 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequencecomprising ATNFSLLKQAGDVEENPGP (SEQ ID NO: 25). A GSG-P2A peptide maycomprise an amino acid sequence comprising GSGATNFSLLKQAGDVEENPGP (SEQID NO: 26) or a sequence having at least 70%, 80%, 90%, 95%, or 99%identity to the amino acid sequence comprising GSGATNFSLLKQAGDVEENPGP(SEQ ID NO: 26).

The disclosure provides a composition comprising the transposon thedisclosure. In certain embodiments, a method introducing the compositionmay further comprise a composition comprising a plasmid comprising asequence encoding a transposase enzyme. The sequence encoding atransposase enzyme may be an mRNA sequence.

Transposons of the disclosure may comprise piggyBac transposons.Transposase enzymes of the disclosure may include piggyBac transposasesor compatible enzymes.

The disclosure provides a vector comprising the CAR of the disclosure.In certain embodiments, the vector is a viral vector. The vector may bea recombinant vector.

Viral vectors of the disclosure may comprise a sequence isolated orderived from a retrovirus, a lentivirus, an adenovirus, anadeno-associated virus or any combination thereof. The viral vector maycomprise a sequence isolated or derived from an adeno-associated virus(AAV). The viral vector may comprise a recombinant AAV (rAAV). Exemplaryadeno-associated viruses and recombinant adeno-associated viruses of thedisclosure comprise two or more inverted terminal repeat (ITR) sequenceslocated in cis next to a sequence encoding a protein scaffold, Centyrinor CARTyrin of the disclosure. Exemplary adeno-associated viruses andrecombinant adeno-associated viruses of the disclosure include, but arenot limited to all serotypes (e.g. AAV1, AAV2, AAV3, AAV4, AAVS, AAV6,AAV7, AAV8, and AAV9). Exemplary adeno-associated viruses andrecombinant adeno-associated viruses of the disclosure include, but arenot limited to, self-complementary AAV (scAAV) and AAV hybridscontaining the genome of one serotype and the capsid of another serotype(e.g. AAV2/5, AAV-DJ and AAV-DJ8). Exemplary adeno-associated virusesand recombinant adeno-associated viruses of the disclosure include, butare not limited to, rAAV-LK03.

Viral vectors of the disclosure may comprise a selection gene. Theselection gene may encode a gene product essential for cell viabilityand survival. The selection gene may encode a gene product essential forcell viability and survival when challenged by selective cell cultureconditions. Selective cell culture conditions may comprise a compoundharmful to cell viability or survival and wherein the gene productconfers resistance to the compound. Exemplary selection genes of thedisclosure may include, but are not limited to, neo (conferringresistance to neomycin), DHFR (encoding Dihydrofolate Reductase andconferring resistance to Methotrexate), TYMS (encoding ThymidylateSynthetase), MGMT (encoding O(6)-methylguanine-DNA methyltransferase),multidrug resistance gene (MDR1), ALDH1 (encoding Aldehyde dehydrogenase1 family, member A1), FRANCF, RAD51C (encoding RAD51 Paralog C), GCS(encoding glucosylceramide synthase), NKX2.2 (encoding NK2 Homeobox 2)or any combination thereof.

Viral vectors of the disclosure may comprise at least one self-cleavingpeptide. In some embodiments, the vector may comprise at least oneself-cleaving peptide and wherein a self-cleaving peptide is locatedbetween a CAR and a selection gene. In some embodiments, the vector maycomprise at least one self-cleaving peptide and wherein a firstself-cleaving peptide is located upstream of a CAR and a secondself-cleaving peptide is located downstream of a CAR. The self-cleavingpeptide may comprise, for example, a T2A peptide, GSG-T2A peptide, anE2A peptide, a GSG-E2A peptide, an F2A peptide, a GSG-F2A peptide, a P2Apeptide, or a GSG-P2A peptide. A T2A peptide may comprise an amino acidsequence comprising EGRGSLLTCGDVEENPGP (SEQ ID NO: 18) or a sequencehaving at least 70%, 80%, 90%, 95%, or 99% identity to the amino acidsequence comprising EGRGSLLTCGDVEENPGP (SEQ ID NO: 18). A GSG-T2Apeptide may comprise an amino acid sequence comprisingGSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 19) or a sequence having at least 70%,80%, 90%, 95%, or 99% identity to the amino acid sequence comprisingGSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 19). A GSG-T2A peptide may comprise anucleic acid sequence comprisingggatctggagagggaaggggaagcctgctgacctgtggagacgtggaggaaaacccaggacca (SEQ IDNO: 20). An E2A peptide may comprise an amino acid sequence comprisingQCTNYALLKLAGDVESNPGP (SEQ ID NO: 21) or a sequence having at least 70%,80%, 90%, 95%, or 99% identity to the amino acid sequence comprisingQCTNYALLKLAGDVESNPGP (SEQ ID NO: 21). A GSG-E2A peptide may comprise anamino acid sequence comprising GSGQCTNYALLKLAGDVESNPGP (SEQ ID NO: 22)or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to theamino acid sequence comprising GSGQCTNYALLKLAGDVESNPGP (SEQ ID NO: 22).An F2A peptide may comprise an amino acid sequence comprisingVKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 23) or a sequence having at least70%, 80%, 90%, 95%, or 99% identity to the amino acid sequencecomprising VKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 23). A GSG-F2A peptide maycomprise an amino acid sequence comprising GSGVKQTLNFDLLKLAGDVESNPGP(SEQ ID NO: 24) or a sequence having at least 70%, 80%, 90%, 95%, or 99%identity to the amino acid sequence comprising GSGVKQTLNFDLLKLAGDVESNPGP(SEQ ID NO: 24). A P2A peptide may comprise an amino acid sequencecomprising ATNFSLLKQAGDVEENPGP (SEQ ID NO: 25) or a sequence having atleast 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequencecomprising ATNFSLLKQAGDVEENPGP (SEQ ID NO: 25). A GSG-P2A peptide maycomprise an amino acid sequence comprising GSGATNFSLLKQAGDVEENPGP (SEQID NO: 26) or a sequence having at least 70%, 80%, 90%, 95%, or 99%identity to the amino acid sequence comprising GSGATNFSLLKQAGDVEENPGP(SEQ ID NO: 26).

The disclosure provides a vector comprising the CAR of the disclosure.In certain embodiments, the vector is an mRNA vector. The vector may bea recombinant mRNA vector. T cells of the disclosure may be expandedprior to contacting the T-cell and the mRNA vector comprising the CAR ofthe disclosure. The T cell comprising the mRNA vector, the modified Tcell, may then be administered to a subject.

The disclosure provides a vector comprising the CAR of the disclosure.In certain embodiments, the vector is a nanoparticle. Exemplarynanoparticle vectors of the disclosure include, but are not limited to,nucleic acids (e.g. RNA, DNA, synthetic nucleotides, modifiednucleotides or any combination thereof), amino acids (L-amino acids,D-amino acids, synthetic amino acids, modified amino acids, or anycombination thereof), polymers (e.g. polymersomes), micelles, lipids(e.g. liposomes), organic molecules (e.g. carbon atoms, sheets, fibers,tubes), inorganic molecules (e.g. calcium phosphate or gold) or anycombination thereof. A nanoparticle vector may be passively or activelytransported across a cell membrane.

Nanoparticle vectors of the disclosure may comprise a selection gene.The selection gene may encode a gene product essential for cellviability and survival. The selection gene may encode a gene productessential for cell viability and survival when challenged by selectivecell culture conditions. Selective cell culture conditions may comprisea compound harmful to cell viability or survival and wherein the geneproduct confers resistance to the compound. Exemplary selection genes ofthe disclosure may include, but are not limited to, neo (conferringresistance to neomycin), DHFR (encoding Dihydrofolate Reductase andconferring resistance to Methotrexate), TYMS (encoding ThymidylateSynthetase), MGMT (encoding O(6)-methylguanine-DNA methyltransferase),multidrug resistance gene (MDR1), ALDH1 (encoding Aldehyde dehydrogenase1 family, member A1), FRANCF, RAD51C (encoding RAD51 Paralog C), GCS(encoding glucosylceramide synthase), NKX2.2 (encoding NK2 Homeobox 2)or any combination thereof.

Nanoparticle vectors of the disclosure may comprise at least oneself-cleaving peptide. In some embodiments, the nanoparticle vector maycomprise at least one self-cleaving peptide and wherein a self-cleavingpeptide is located between a CAR and the nanoparticle. In someembodiments, the nanoparticle vector may comprise at least oneself-cleaving peptide and wherein a first self-cleaving peptide islocated upstream of a CAR and a second self-cleaving peptide is locateddownstream of a CAR. In some embodiments, the nanoparticle vector maycomprise at least one self-cleaving peptide and wherein a firstself-cleaving peptide is located between a CAR and the nanoparticle anda second self-cleaving peptide is located downstream of the CAR. In someembodiments, the nanoparticle vector may comprise at least oneself-cleaving peptide and wherein a first self-cleaving peptide islocated between a CAR and the nanoparticle and a second self-cleavingpeptide is located downstream of the CAR, for example, between the CARand a selection gene. The self-cleaving peptide may comprise, forexample, a T2A peptide, GSG-T2A peptide, an E2A peptide, a GSG-E2Apeptide, an F2A peptide, a GSG-F2A peptide, a P2A peptide, or a GSG-P2Apeptide. A T2A peptide may comprise an amino acid sequence comprisingEGRGSLLTCGDVEENPGP (SEQ ID NO: 18) or a sequence having at least 70%,80%, 90%, 95%, or 99% identity to the amino acid sequence comprisingEGRGSLLTCGDVEENPGP (SEQ ID NO: 18). A GSG-T2A peptide may comprise anamino acid sequence comprising GSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 19) ora sequence having at least 70%, 80%, 90%, 95%, or 99% identity to theamino acid sequence comprising GSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 19). AGSG-T2A peptide may comprise a nucleic acid sequence comprisingggatctggagagggaaggggaagcctgctgacctgtggagacgtggaggaaaacccaggacca (SEQ IDNO: 20). An E2A peptide may comprise an amino acid sequence comprisingQCTNYALLKLAGDVESNPGP (SEQ ID NO: 21) or a sequence having at least 70%,80%, 90%, 95%, or 99% identity to the amino acid sequence comprisingQCTNYALLKLAGDVESNPGP (SEQ ID NO: 21). A GSG-E2A peptide may comprise anamino acid sequence comprising GSGQCTNYALLKLAGDVESNPGP (SEQ ID NO: 22)or a sequence having at least 70%, 80%, 90%, 95%, or 99% identity to theamino acid sequence comprising GSGQCTNYALLKLAGDVESNPGP (SEQ ID NO: 22).An F2A peptide may comprise an amino acid sequence comprisingVKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 23) or a sequence having at least70%, 80%, 90%, 95%, or 99% identity to the amino acid sequencecomprising VKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 23). A GSG-F2A peptide maycomprise an amino acid sequence comprising GSGVKQTLNFDLLKLAGDVESNPGP(SEQ ID NO: 24) or a sequence having at least 70%, 80%, 90%, 95%, or 99%identity to the amino acid sequence comprising GSGVKQTLNFDLLKLAGDVESNPGP(SEQ ID NO: 24). A P2A peptide may comprise an amino acid sequencecomprising ATNFSLLKQAGDVEENPGP (SEQ ID NO: 25) or a sequence having atleast 70%, 80%, 90%, 95%, or 99% identity to the amino acid sequencecomprising ATNFSLLKQAGDVEENPGP (SEQ ID NO: 25). A GSG-P2A peptide maycomprise an amino acid sequence comprising GSGATNFSLLKQAGDVEENPGP (SEQID NO: 26) or a sequence having at least 70%, 80%, 90%, 95%, or 99%identity to the amino acid sequence comprising GSGATNFSLLKQAGDVEENPGP(SEQ ID NO: 26).

The disclosure provides a composition comprising a vector of thedisclosure.

Scaffold Proteins

A Centyrin is one example of a protein scaffold of the disclosure. Anantigen recognition region of a CAR of the disclosure may comprise atleast one protein scaffold.

Protein scaffolds of the disclosure may be derived from a fibronectintype III (FN3) repeat protein, encoding or complementary nucleic acids,vectors, host cells, compositions, combinations, formulations, devices,and methods of making and using them. In a preferred embodiment, theprotein scaffold is comprised of a consensus sequence of multiple FN3domains from human Tenascin-C (hereinafter “Tenascin”). In a furtherpreferred embodiment, the protein scaffold of the present invention is aconsensus sequence of 15 FN3 domains. The protein scaffolds of thedisclosure can be designed to bind various molecules, for example, acellular target protein. In a preferred embodiment, the proteinscaffolds of the disclosure can be designed to bind an epitope of a wildtype and/or variant form of an antigen.

Protein scaffolds of the disclosure may include additional molecules ormoieties, for example, the Fc region of an antibody, albumin bindingdomain, or other moiety influencing half-life. In further embodiments,the protein scaffolds of the disclosure may be bound to a nucleic acidmolecule that may encode the protein scaffold.

The disclosure provides at least one method for expressing at least oneprotein scaffold based on a consensus sequence of multiple FN3 domains,in a host cell, comprising culturing a host cell as described hereinunder conditions wherein at least one protein scaffold is expressed indetectable and/or recoverable amounts.

The disclosure provides at least one composition comprising (a) aprotein scaffold based on a consensus sequence of multiple FN3 domainsand/or encoding nucleic acid as described herein; and (b) a suitableand/or pharmaceutically acceptable carrier or diluent.

The disclosure provides a method of generating libraries of a proteinscaffold based on a fibronectin type III (FN3) repeat protein,preferably, a consensus sequence of multiple FN3 domains and, morepreferably, a consensus sequence of multiple FN3 domains from humanTenascin. The library is formed by making successive generations ofscaffolds by altering (by mutation) the amino acids or the number ofamino acids in the molecules in particular positions in portions of thescaffold, e.g., loop regions. Libraries can be generated by altering theamino acid composition of a single loop or the simultaneous alterationof multiple loops or additional positions of the scaffold molecule. Theloops that are altered can be lengthened or shortened accordingly. Suchlibraries can be generated to include all possible amino acids at eachposition, or a designed subset of amino acids. The library members canbe used for screening by display, such as in vitro or CIS display (DNA,RNA, ribosome display, etc.), yeast, bacterial, and phage display.

Protein scaffolds of the disclosure provide enhanced biophysicalproperties, such as stability under reducing conditions and solubilityat high concentrations; they may be expressed and folded in prokaryoticsystems, such as E. coli, in eukaryotic systems, such as yeast, and inin vitro transcription/translation systems, such as the rabbitreticulocyte lysate system.

The disclosure provides an isolated, recombinant and/or syntheticprotein scaffold based on a consensus sequence of fibronectin type III(FN3) repeat protein, including, without limitation, mammalian-derivedscaffold, as well as compositions and encoding nucleic acid moleculescomprising at least one polynucleotide encoding protein scaffold basedon the consensus FN3 sequence. The disclosure further includes, but isnot limited to, methods of making and using such nucleic acids andprotein scaffolds, including diagnostic and therapeutic compositions,methods and devices.

The protein scaffolds of the disclosure offer advantages overconventional therapeutics, such as ability to administer locally,orally, or cross the blood-brain barrier, ability to express in E. Coliallowing for increased expression of protein as a function of resourcesversus mammalian cell expression ability to be engineered intobispecific or tandem molecules that bind to multiple targets or multipleepitopes of the same target, ability to be conjugated to drugs,polymers, and probes, ability to be formulated to high concentrations,and the ability of such molecules to effectively penetrate diseasedtissues and tumors.

Moreover, the protein scaffolds possess many of the properties ofantibodies in relation to their fold that mimics the variable region ofan antibody. This orientation enables the FN3 loops to be exposedsimilar to antibody complementarity determining regions (CDRs). Theyshould be able to bind to cellular targets and the loops can be altered,e.g., affinity matured, to improve certain binding or relatedproperties.

Three of the six loops of the protein scaffold of the disclosurecorrespond topologically to the complementarity determining regions(CDRs 1-3), i.e., antigen-binding regions, of an antibody, while theremaining three loops are surface exposed in a manner similar toantibody CDRs. These loops span at or about residues 13-16, 22-28,38-43, 51-54, 60-64, and 75-81 of SEQ ID NO: 1. Preferably, the loopregions at or about residues 22-28, 51-54, and 75-81 are altered forbinding specificity and affinity. One or more of these loop regions arerandomized with other loop regions and/or other strands maintainingtheir sequence as backbone portions to populate a library and potentbinders can be selected from the library having high affinity for aparticular protein target. One or more of the loop regions can interactwith a target protein similar to an antibody CDR interaction with theprotein.

Scaffolds of the disclosure may comprise a single chain antibody (e.g. ascFv). Single chain antibodies of the disclosure may comprise threelight chain and three heavy chain CDRs of an antibody. In certainembodiments, the single chain antibodies of the disclosure comprisethree light chain and three heavy chain CDRs of an antibody, wherein thecomplementarity-determining regions (CDRs) of the single chain antibodyare human sequences. The disclosure provides a chimeric antigen receptor(CAR) comprising: (a) an ectodomain comprising an antigen recognitionregion, wherein the antigen recognition region comprises at least onesingle chain antibody (e.g. a scFv); (b) a transmembrane domain, and (c)an endodomain comprising at least one costimulatory domain. In certainembodiments, the antigen recognition region may comprise two singlechain antibodies (e.g. two scFvs) to produce a bi-specific or tandemCAR. In certain embodiments, the antigen recognition region may comprisethree single chain antibodies (e.g. three scFvs) to produce atri-specific CAR. In certain embodiments, the ectodomain may furthercomprise a signal peptide. Alternatively, or in addition, in certainembodiments, the ectodomain may further comprise a hinge between theantigen recognition region and the transmembrane domain.

Scaffolds of the disclosure may comprise a sequence comprising one ormore fragments of an antibody (e.g. a VHH). Sequence comprising one ormore fragments of an antibody of the disclosure may comprise two heavychain variable regions of an antibody. In certain embodiments, thesequence comprises two heavy chain variable regions of an antibody,wherein the complementarity-determining regions (CDRs) of the VHH arehuman sequences. Scaffolds of the disclosure may comprise a sequencecomprising one or more fragments of an antibody (e.g. a VHH). Thedisclosure provides a chimeric antigen receptor (CAR) comprising: (a) anectodomain comprising an antigen recognition region, wherein the antigenrecognition region comprises at least one a sequence comprising one ormore fragments of an antibody (e.g. a VHH); (b) a transmembrane domain,and (c) an endodomain comprising at least one costimulatory domain. Incertain embodiments, the antigen recognition region may comprise twosequences comprising one or more fragments of an antibody (e.g. twoVHHs) to produce a bi-specific or tandem CAR. In certain embodiments,the antigen recognition region may comprise three sequences comprisingone or more fragments of an antibody (e.g. three VHHs) to produce atri-specific CAR. In certain embodiments, the ectodomain may furthercomprise a signal peptide. Alternatively, or in addition, in certainembodiments, the ectodomain may further comprise a hinge between theantigen recognition region and the transmembrane domain.

Scaffolds of the disclosure may comprise an antibody mimetic.

The term “antibody mimetic” is intended to describe an organic compoundthat specifically binds a target sequence and has a structure distinctfrom a naturally-occurring antibody. Antibody mimetics may comprise aprotein, a nucleic acid, or a small molecule. The target sequence towhich an antibody mimetic of the disclosure specifically binds may be anantigen. Antibody mimetics may provide superior properties overantibodies including, but not limited to, superior solubility, tissuepenetration, stability towards heat and enzymes (e.g. resistance toenzymatic degradation), and lower production costs. Exemplary antibodymimetics include, but are not limited to, an affibody, an afflilin, anaffimer, an affitin, an alphabody, an anticalin, and avimer (also knownas avidity multimer), a DARPin (Designed Ankyrin Repeat Protein), aFynomer, a Kunitz domain peptide, and a monobody.

Affibody molecules of the disclosure comprise a protein scaffoldcomprising or consisting of one or more alpha helix without anydisulfide bridges. Preferably, affibody molecules of the disclosurecomprise or consist of three alpha helices. For example, an affibodymolecule of the disclosure may comprise an immunoglobulin bindingdomain. An affibody molecule of the disclosure may comprise the Z domainof protein A.

Affilin molecules of the disclosure comprise a protein scaffold producedby modification of exposed amino acids of, for example, either gamma-Bcrystallin or ubiquitin. Affilin molecules functionally mimic anantibody's affinity to antigen, but do not structurally mimic anantibody. In any protein scaffold used to make an affilin, those aminoacids that are accessible to solvent or possible binding partners in aproperly-folded protein molecule are considered exposed amino acids. Anyone or more of these exposed amino acids may be modified to specificallybind to a target sequence or antigen.

Affimer molecules of the disclosure comprise a protein scaffoldcomprising a highly stable protein engineered to display peptide loopsthat provide a high affinity binding site for a specific targetsequence. Exemplary affimer molecules of the disclosure comprise aprotein scaffold based upon a cystatin protein or tertiary structurethereof. Exemplary affimer molecules of the disclosure may share acommon tertiary structure of comprising an alpha-helix lying on top ofan anti-parallel beta-sheet.

Affitin molecules of the disclosure comprise an artificial proteinscaffold, the structure of which may be derived, for example, from a DNAbinding protein (e.g. the DNA binding protein Sac7d). Affitins of thedisclosure selectively bind a target sequence, which may be the entiretyor part of an antigen. Exemplary affitins of the disclosure aremanufactured by randomizing one or more amino acid sequences on thebinding surface of a DNA binding protein and subjecting the resultantprotein to ribosome display and selection. Target sequences of affitinsof the disclosure may be found, for example, in the genome or on thesurface of a peptide, protein, virus, or bacteria. In certainembodiments of the disclosure, an affitin molecule may be used as aspecific inhibitor of an enzyme. Affitin molecules of the disclosure mayinclude heat-resistant proteins or derivatives thereof.

Alphabody molecules of the disclosure may also be referred to asCell-Penetrating Alphabodies (CPAB). Alphabody molecules of thedisclosure comprise small proteins (typically of less than 10 kDa) thatbind to a variety of target sequences (including antigens). Alphabodymolecules are capable of reaching and binding to intracellular targetsequences. Structurally, alphabody molecules of the disclosure comprisean artificial sequence forming single chain alpha helix (similar tonaturally occurring coiled-coil structures). Alphabody molecules of thedisclosure may comprise a protein scaffold comprising one or more aminoacids that are modified to specifically bind target proteins. Regardlessof the binding specificity of the molecule, alphabody molecules of thedisclosure maintain correct folding and thermostability.

Anticalin molecules of the disclosure comprise artificial proteins thatbind to target sequences or sites in either proteins or small molecules.Anticalin molecules of the disclosure may comprise an artificial proteinderived from a human lipocalin. Anticalin molecules of the disclosuremay be used in place of, for example, monoclonal antibodies or fragmentsthereof. Anticalin molecules may demonstrate superior tissue penetrationand thermostability than monoclonal antibodies or fragments thereof.Exemplary anticalin molecules of the disclosure may comprise about 180amino acids, having a mass of approximately 20 kDa. Structurally,anticalin molecules of the disclosure comprise a barrel structurecomprising antiparallel beta-strands pairwise connected by loops and anattached alpha helix. In preferred embodiments, anticalin molecules ofthe disclosure comprise a barrel structure comprising eight antiparallelbeta-strands pairwise connected by loops and an attached alpha helix.

Avimer molecules of the disclosure comprise an artificial protein thatspecifically binds to a target sequence (which may also be an antigen).Avimers of the disclosure may recognize multiple binding sites withinthe same target or within distinct targets. When an avimer of thedisclosure recognize more than one target, the avimer mimics function ofa bi-specific antibody. The artificial protein avimer may comprise twoor more peptide sequences of approximately 30-35 amino acids each. Thesepeptides may be connected via one or more linker peptides. Amino acidsequences of one or more of the peptides of the avimer may be derivedfrom an A domain of a membrane receptor. Avimers have a rigid structurethat may optionally comprise disulfide bonds and/or calcium. Avimers ofthe disclosure may demonstrate greater heat stability compared to anantibody.

DARPins (Designed Ankyrin Repeat Proteins) of the disclosure comprisegenetically-engineered, recombinant, or chimeric proteins having highspecificity and high affinity for a target sequence. In certainembodiments, DARPins of the disclosure are derived from ankyrin proteinsand, optionally, comprise at least three repeat motifs (also referred toas repetitive structural units) of the ankyrin protein. Ankyrin proteinsmediate high-affinity protein-protein interactions. DARPins of thedisclosure comprise a large target interaction surface.

Fynomers of the disclosure comprise small binding proteins (about 7 kDa)derived from the human Fyn SH3 domain and engineered to bind to targetsequences and molecules with equal affinity and equal specificity as anantibody.

Kunitz domain peptides of the disclosure comprise a protein scaffoldcomprising a Kunitz domain. Kunitz domains comprise an active site forinhibiting protease activity. Structurally, Kunitz domains of thedisclosure comprise a disulfide-rich alpha+beta fold. This structure isexemplified by the bovine pancreatic trypsin inhibitor. Kunitz domainpeptides recognize specific protein structures and serve as competitiveprotease inhibitors. Kunitz domains of the disclosure may compriseEcallantide (derived from a human lipoprotein-associated coagulationinhibitor (LACI)).

Monobodies of the disclosure are small proteins (comprising about 94amino acids and having a mass of about 10 kDa) comparable in size to asingle chain antibody. These genetically engineered proteinsspecifically bind target sequences including antigens. Monobodies of thedisclosure may specifically target one or more distinct proteins ortarget sequences. In preferred embodiments, monobodies of the disclosurecomprise a protein scaffold mimicking the structure of humanfibronectin, and more preferably, mimicking the structure of the tenthextracellular type III domain of fibronectin. The tenth extracellulartype III domain of fibronectin, as well as a monobody mimetic thereof,contains seven beta sheets forming a barrel and three exposed loops oneach side corresponding to the three complementarity determining regions(CDRs) of an antibody. In contrast to the structure of the variabledomain of an antibody, a monobody lacks any binding site for metal ionsas well as a central disulfide bond. Multispecific monobodies may beoptimized by modifying the loops BC and FG. Monobodies of the disclosuremay comprise an adnectin.

Production and Generation of Scaffold Proteins

At least one scaffold protein of the disclosure can be optionallyproduced by a cell line, a mixed cell line, an immortalized cell orclonal population of immortalized cells, as well known in the art. See,e.g., Ausubel, et al., ed., Current Protocols in Molecular Biology, JohnWiley & Sons, Inc., NY, N.Y. (1987-2001); Sambrook, et al., MolecularCloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor, N.Y.(1989); Harlow and Lane, Antibodies, a Laboratory Manual, Cold SpringHarbor, N.Y. (1989); Colligan, et al., eds., Current Protocols inImmunology, John Wiley & Sons, Inc., NY (1994-2001); Colligan et al.,Current Protocols in Protein Science, John Wiley & Sons, NY, N.Y.,(1997-2001).

Amino acids from a scaffold protein can be altered, added and/or deletedto reduce immunogenicity or reduce, enhance or modify binding, affinity,on-rate, off-rate, avidity, specificity, half-life, stability,solubility or any other suitable characteristic, as known in the art.

Optionally, scaffold proteins can be engineered with retention of highaffinity for the antigen and other favorable biological properties. Toachieve this goal, the scaffold proteins can be optionally prepared by aprocess of analysis of the parental sequences and various conceptualengineered products using three-dimensional models of the parental andengineered sequences. Three-dimensional models are commonly availableand are familiar to those skilled in the art. Computer programs areavailable which illustrate and display probable three-dimensionalconformational structures of selected candidate sequences and canmeasure possible immunogenicity (e.g., Immunofilter program of Xencor,Inc. of Monrovia, Calif.). Inspection of these displays permits analysisof the likely role of the residues in the functioning of the candidatesequence, i.e., the analysis of residues that influence the ability ofthe candidate scaffold protein to bind its antigen. In this way,residues can be selected and combined from the parent and referencesequences so that the desired characteristic, such as affinity for thetarget antigen(s), is achieved. Alternatively, or in addition to, theabove procedures, other suitable methods of engineering can be used.

piggyBac Transposon System

The methods of the disclosure produce a modified T_(SCM) of thedisclosure regardless of the method used for introducing an antigenreceptor into a primary human T cell of the disclosure. The methods ofthe disclosure produce a modified T_(SCM) of the disclosure with greaterefficacy and/or a greater abundance, proportion, yield of modified−T_(SCM) of the disclosure when the antigen receptor or the therapeuticprotein of the disclosure is introduced to the primary human T cellusing the piggyBac transposon system. A piggyBac transposon system ofthe disclosure may comprise a piggyBac transposon comprising an antigenreceptor of the disclosure. Preferably, the primary human T cellcontacts a piggyBac transposon comprising an antigen receptor of thedisclosure and a transposase of the disclosure simultaneously (or invery close temporal proximity, e.g. the primary human T cell, thetransposon and the transposase are contained in the same container (suchas a cuvette) prior to introduction of the transposon and transposaseinto the cell—however they would not be permitted to interact in theabsence of the cell. Preferably, the primary human T cell contacts apiggyBac transposon comprising an antigen receptor of the disclosure anda Super piggyBac™ (SPB) transposase of the disclosure simultaneouslyprior to introduction of the transposon and transposase into the cell.In certain preferred embodiments, the Super piggyBac™ (SPB) transposaseis an mRNA sequence encoding the Super piggyBac™ (SPB) transposase.

Additional disclosure regarding piggyBac transposons and Super piggyBac™(SPB) transposases may be found in International Patent Publication WO2010/099296, U.S. Pat. Nos. 8,399,643, 9,546,382, 6,218,185, 6,551,825,6,962,810, and 7,105,343, the contents of which are each hereinincorporated by reference in their entireties.

The disclosure provides methods of introducing a polynucleotideconstruct comprising a DNA sequence into a host cell. Preferably, theintroducing steps are mediated by the piggyBac transposon system.

In certain embodiments of the methods of the disclosure, the transposonis a plasmid DNA transposon with a sequence encoding the antigenreceptor or the therapeutic protein flanked by two cis-regulatoryinsulator elements. In certain embodiments, the transposon is a piggyBactransposon. In certain embodiments, and, in particular, thoseembodiments wherein the transposon is a piggyBac transposon, thetransposase is a piggyBac™ or a Super piggyBac™ (SPB) transposase. Incertain embodiments, and, in particular, those embodiments wherein thetransposase is a Super piggyBac™ (SPB) transposase, the sequenceencoding the transposase is an mRNA sequence.

In certain embodiments of the methods of the disclosure, the transposaseenzyme is a piggyBac™ (PB) transposase enzyme. The piggyBac (PB)transposase enzyme may comprise or consist of an amino acid sequence atleast 75%, 80%, 85%, 90%, 95%, 99% or any percentage in betweenidentical to:

(SEQ ID NO: 4)  1 MGSSLDDEHI LSALLQSDDE LVGEDSDSEI SDHVSEDDVQ SDTEEAFIDE VHEVQPTSSG 61 SEILDEQNVI EQPGSSLASN RILTLPQRTI RGKNKHCWST SKSTRRSRVS ALNIVRSQRG121 PTRMCRNIYD PLLCFKLFFT DEIISEIVKW TNAEISLKRR ESMTGATFRD TNEDEIYAFF181 GILVMTAVRK DNHMSTDDLF DRSLSMVYVS VMSRDRFDFL IRCLRMDDKS IRPTLRENDV241 FTPVRKIWDL FIHQCIQNYT PGAHLTIDEQ LLGFRGRCPF RMYIPNKPSK YGIKILMMCD301 SGYKYMINGM PYLGRGTQTN GVPLGEYYVK ELSKPVHGSC RNITCDNWFT SIPLAKNLLQ361 EPYKLTIVGT VRSNKREIPE VLKNSRSRPV GTSMFCFDGP LTLVSYKPKP AKMVYLLSSC421 DEDASINEST GKPQMVMYYN QTKGGVDTLD QMCSVMTCSR KTNRWPMALL YGMINIACIN481 SFIIYSHNVS SKGEKVQSRK KFMRNLYMSL TSSFMRKRLE APTLKRYLRD NISNILPNEV541 PGTSDDSTEE PVMKKRTYCT YCPSKIRRKA NASCKKCKKV ICREHNIDMC QSCF.

In certain embodiments of the methods of the disclosure, the transposaseenzyme is a piggyBac™ (PB) transposase enzyme that comprises or consistsof an amino acid sequence having an amino acid substitution at one ormore of positions 30, 165, 282, or 538 of the sequence:

(SEQ ID NO: 4)  1 MGSSLDDEHI LSALLQSDDE LVGEDSDSEI SDHVSEDDVQ SDTEEAFIDE VHEVQPTSSG 61 SEILDEQNVI EQPGSSLASN RILTLPQRTI RGKNKHCWST SKSTRRSRVS ALNIVRSQRG121 PTRMCRNIYD PLLCFKLFFT DEIISEIVKW TNAEISLKRR ESMTGATFRD TNEDEIYAFF181 GILVMTAVRK DNHMSTDDLF DRSLSMVYVS VMSRDRFDFL IRCLRMDDKS IRPTLRENDV241 FTPVRKIWDL FIHQCIQNYT PGAHLTIDEQ LLGFRGRCPF RMYIPNKPSK YGIKILMMCD301 SGYKYMINGM PYLGRGTQTN GVPLGEYYVK ELSKPVHGSC RNITCDNWFT SIPLAKNLLQ361 EPYKLTIVGT VRSNKREIPE VLKNSRSRPV GTSMFCFDGP LTLVSYKPKP AKMVYLLSSC421 DEDASINEST GKPQMVMYYN QTKGGVDTLD QMCSVMTCSR KTNRWPMALL YGMINIACIN481 SFIIYSHNVS SKGEKVQSRK KFMRNLYMSL TSSFMRKRLE APTLKRYLRD NISNILPNEV541 PGTSDDSTEE PVMKKRTYCT YCPSKIRRKA NASCKKCKKV ICREHNIDMC QSCF.

In certain embodiments, the transposase enzyme is a piggyBac™ (PB)transposase enzyme that comprises or consists of an amino acid sequencehaving an amino acid substitution at two or more of positions 30, 165,282, or 538 of the sequence of SEQ ID NO: 4. In certain embodiments, thetransposase enzyme is a piggyBac™ (PB) transposase enzyme that comprisesor consists of an amino acid sequence having an amino acid substitutionat three or more of positions 30, 165, 282, or 538 of the sequence ofSEQ ID NO: 4. In certain embodiments, the transposase enzyme is apiggyBac™ (PB) transposase enzyme that comprises or consists of an aminoacid sequence having an amino acid substitution at each of the followingpositions 30, 165, 282, and 538 of the sequence of SEQ ID NO: 4. Incertain embodiments, the amino acid substitution at position 30 of thesequence of SEQ ID NO: 4 is a substitution of a valine (V) for anisoleucine (I). In certain embodiments, the amino acid substitution atposition 165 of the sequence of SEQ ID NO: 4 is a substitution of aserine (S) for a glycine (G). In certain embodiments, the amino acidsubstitution at position 282 of the sequence of SEQ ID NO: 4 is asubstitution of a valine (V) for a methionine (M). In certainembodiments, the amino acid substitution at position 538 of the sequenceof SEQ ID NO: 4 is a substitution of a lysine (K) for an asparagine (N).

In certain embodiments of the methods of the disclosure, the transposaseenzyme is a Super piggyBac™ (SPB) transposase enzyme. In certainembodiments, the Super piggyBac™ (SPB) transposase enzymes of thedisclosure may comprise or consist of the amino acid sequence of thesequence of SEQ ID NO: 4 wherein the amino acid substitution at position30 is a substitution of a valine (V) for an isoleucine (I), the aminoacid substitution at position 165 is a substitution of a serine (S) fora glycine (G), the amino acid substitution at position 282 is asubstitution of a valine (V) for a methionine (M), and the amino acidsubstitution at position 538 is a substitution of a lysine (K) for anasparagine (N). In certain embodiments, the Super piggyBac™ (SPB)transposase enzyme may comprise or consist of an amino acid sequence atleast 75%, 80%, 85%, 90%, 95%, 99% or any percentage in betweenidentical to:

(SEQ ID NO: 5)  1 MGSSLDDEHI LSALLQSDDE LVGEDSDSEV SDHVSEDDVQ SDTEEAFIDE VHEVQPTSSG 61 SEILDEQNVI EQPGSSLASN RILTLPQRTI RGKNKHCWST SKSTRRSRVS ALNIVRSQRG121 PTRMCRNIYD PLLCFKLFFT DEIISEIVKW TNAEISLKRR ESMTSATFRD TNEDEIYAFF181 GILVMTAVRK DNHMSTDDLF DRSLSMVYVS VMSRDRFDFL IRCLRMDDKS IRPTLRENDV241 FTPVRKIWDL FIHQCIQNYT PGAHLTIDEQ LLGFRGRCPF RVYIPNKPSK YGIKILMMCD301 SGTKYMINGM PYLGRGTQTN GVPLGEYYVK ELSKPVHGSC RNITCDNWFT SIPLAKNLLQ361 EPYKLTIVGT VRSNKREIPE VLKNSRSRPV GTSMFCFDGP LTLVSYKPKP AKMVYLLSSC421 DEDASINEST GKPQMVMYYN QTKGGVDTLD QMCSVMTCSR KTNRWPMALL YGMINIACIN481 SFIIYSHNVS SKGEKVQSRK KFMRNLYMSL TSSFMRKRLE APTLKRYLRD NISNILPKEV541 PGTSDDSTEE PVMKKRTYCT YCPSKIRRKA NASCKKCKKV ICREHNIDMC QSCF.

In certain embodiments of the methods of the disclosure, including thoseembodiments wherein the transposase comprises the above-describedmutations at positions 30, 165, 282 and/or 538, the piggyBac™ or SuperpiggyBac™ transposase enzyme may further comprise an amino acidsubstitution at one or more of positions 3, 46, 82, 103, 119, 125, 177,180, 185, 187, 200, 207, 209, 226, 235, 240, 241, 243, 258, 296, 298,311, 315, 319, 327, 328, 340, 421, 436, 456, 470, 486, 503, 552, 570 and591 of the sequence of SEQ ID NO: 4 or SEQ ID NO: 5. In certainembodiments, including those embodiments wherein the transposasecomprises the above-described mutations at positions 30, 165, 282 and/or538, the piggyBac™ or Super piggyBac™ transposase enzyme may furthercomprise an amino acid substitution at one or more of positions 46, 119,125, 177, 180, 185, 187, 200, 207, 209, 226, 235, 240, 241, 243, 296,298, 311, 315, 319, 327, 328, 340, 421, 436, 456, 470, 485, 503, 552 and570. In certain embodiments, the amino acid substitution at position 3of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of an asparagine (N)for a serine (S). In certain embodiments, the amino acid substitution atposition 46 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of aserine (S) for an alanine (A). In certain embodiments, the amino acidsubstitution at position 46 of SEQ ID NO: 4 or SEQ ID NO: 5 is asubstitution of a threonine (T) for an alanine (A). In certainembodiments, the amino acid substitution at position 82 of SEQ ID NO: 4or SEQ ID NO: 5 is a substitution of a tryptophan (W) for an isoleucine(I). In certain embodiments, the amino acid substitution at position 103of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a proline (P) for aserine (S). In certain embodiments, the amino acid substitution atposition 119 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of aproline (P) for an arginine (R). In certain embodiments, the amino acidsubstitution at position 125 of SEQ ID NO: 4 or SEQ ID NO: 5 is asubstitution of an alanine (A) a cysteine (C). In certain embodiments,the amino acid substitution at position 125 of SEQ ID NO: 4 or SEQ IDNO: 5 is a substitution of a leucine (L) for a cysteine (C). In certainembodiments, the amino acid substitution at position 177 of SEQ ID NO: 4or SEQ ID NO: 5 is a substitution of a lysine (K) for a tyrosine (Y). Incertain embodiments, the amino acid substitution at position 177 of SEQID NO: 4 or SEQ ID NO: 5 is a substitution of a histidine (H) for atyrosine (Y). In certain embodiments, the amino acid substitution atposition 180 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of aleucine (L) for a phenylalanine (F). In certain embodiments, the aminoacid substitution at position 180 of SEQ ID NO: 4 or SEQ ID NO: 5 is asubstitution of an isoleucine (I) for a phenylalanine (F). In certainembodiments, the amino acid substitution at position 180 of SEQ ID NO: 4or SEQ ID NO: 5 is a substitution of a valine (V) for a phenylalanine(F). In certain embodiments, the amino acid substitution at position 185of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a leucine (L) for amethionine (M). In certain embodiments, the amino acid substitution atposition 187 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of aglycine (G) for an alanine (A). In certain embodiments, the amino acidsubstitution at position 200 of SEQ ID NO: 4 or SEQ ID NO: 5 is asubstitution of a tryptophan (W) for a phenylalanine (F). In certainembodiments, the amino acid substitution at position 207 of SEQ ID NO: 4or SEQ ID NO: 5 is a substitution of a proline (P) for a valine (V). Incertain embodiments, the amino acid substitution at position 209 of SEQID NO: 4 or SEQ ID NO: 5 is a substitution of a phenylalanine (F) for avaline (V). In certain embodiments, the amino acid substitution atposition 226 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of aphenylalanine (F) for a methionine (M). In certain embodiments, theamino acid substitution at position 235 of SEQ ID NO: 4 or SEQ ID NO: 5is a substitution of an arginine (R) for a leucine (L). In certainembodiments, the amino acid substitution at position 240 of SEQ ID NO: 4or SEQ ID NO: 5 is a substitution of a lysine (K) for a valine (V). Incertain embodiments, the amino acid substitution at position 241 of SEQID NO: 4 or SEQ ID NO: 5 is a substitution of a leucine (L) for aphenylalanine (F). In certain embodiments, the amino acid substitutionat position 243 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of alysine (K) for a proline (P). In certain embodiments, the amino acidsubstitution at position 258 of SEQ ID NO: 4 or SEQ ID NO: 5 is asubstitution of a serine (S) for an asparagine (N). In certainembodiments, the amino acid substitution at position 296 of SEQ ID NO: 4or SEQ ID NO: 5 is a substitution of a tryptophan (W) for a leucine (L).In certain embodiments, the amino acid substitution at position 296 ofSEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a tyrosine (Y) for aleucine (L). In certain embodiments, the amino acid substitution atposition 296 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of aphenylalanine (F) for a leucine (L). In certain embodiments, the aminoacid substitution at position 298 of SEQ ID NO: 4 or SEQ ID NO: 5 is asubstitution of a leucine (L) for a methionine (M). In certainembodiments, the amino acid substitution at position 298 of SEQ ID NO: 4or SEQ ID NO: 5 is a substitution of an alanine (A) for a methionine(M). In certain embodiments, the amino acid substitution at position 298of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a valine (V) for amethionine (M). In certain embodiments, the amino acid substitution atposition 311 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of anisoleucine (I) for a proline (P). In certain embodiments, the amino acidsubstitution at position 311 of SEQ ID NO: 4 or SEQ ID NO: 5 is asubstitution of a valine for a proline (P). In certain embodiments, theamino acid substitution at position 315 of SEQ ID NO: 4 or SEQ ID NO: 5is a substitution of a lysine (K) for an arginine (R). In certainembodiments, the amino acid substitution at position 319 of SEQ ID NO: 4or SEQ ID NO: 5 is a substitution of a glycine (G) for a threonine (T).In certain embodiments, the amino acid substitution at position 327 ofSEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of an arginine (R) for atyrosine (Y). In certain embodiments, the amino acid substitution atposition 328 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of avaline (V) for a tyrosine (Y). In certain embodiments, the amino acidsubstitution at position 340 of SEQ ID NO: 4 or SEQ ID NO: 5 is asubstitution of a glycine (G) for a cysteine (C). In certainembodiments, the amino acid substitution at position 340 of SEQ ID NO: 4or SEQ ID NO: 5 is a substitution of a leucine (L) for a cysteine (C).In certain embodiments, the amino acid substitution at position 421 ofSEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a histidine (H) forthe aspartic acid (D). In certain embodiments, the amino acidsubstitution at position 436 of SEQ ID NO: 4 or SEQ ID NO: 5 is asubstitution of an isoleucine (I) for a valine (V). In certainembodiments, the amino acid substitution at position 456 of SEQ ID NO: 4or SEQ ID NO: 5 is a substitution of a tyrosine (Y) for a methionine(M). In certain embodiments, the amino acid substitution at position 470of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a phenylalanine (F)for a leucine (L). In certain embodiments, the amino acid substitutionat position 485 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of alysine (K) for a serine (S). In certain embodiments, the amino acidsubstitution at position 503 of SEQ ID NO: 4 or SEQ ID NO: 5 is asubstitution of a leucine (L) for a methionine (M). In certainembodiments, the amino acid substitution at position 503 of SEQ ID NO: 4or SEQ ID NO: 5 is a substitution of an isoleucine (I) for a methionine(M). In certain embodiments, the amino acid substitution at position 552of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of a lysine (K) for avaline (V). In certain embodiments, the amino acid substitution atposition 570 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of athreonine (T) for an alanine (A). In certain embodiments, the amino acidsubstitution at position 591 of SEQ ID NO: 4 or SEQ ID NO: 5 is asubstitution of a proline (P) for a glutamine (Q). In certainembodiments, the amino acid substitution at position 591 of SEQ ID NO: 4or SEQ ID NO: 5 is a substitution of an arginine (R) for a glutamine(Q).

In certain embodiments of the methods of the disclosure, including thoseembodiments wherein the transposase comprises the above-describedmutations at positions 30, 165, 282 and/or 538, the piggyBac™transposase enzyme may comprise or the Super piggyBac™ transposaseenzyme may further comprise an amino acid substitution at one or more ofpositions 103, 194, 372, 375, 450, 509 and 570 of the sequence of SEQ IDNO: 4 or SEQ ID NO: 5. In certain embodiments of the methods of thedisclosure, including those embodiments wherein the transposasecomprises the above-described mutations at positions 30, 165, 282 and/or538, the piggyBac™ transposase enzyme may comprise or the SuperpiggyBac™ transposase enzyme may further comprise an amino acidsubstitution at two, three, four, five, six or more of positions 103,194, 372, 375, 450, 509 and 570 of the sequence of SEQ ID NO: 4 or SEQID NO: 5. In certain embodiments, including those embodiments whereinthe transposase comprises the above-described mutations at positions 30,165, 282 and/or 538, the piggyBac™ transposase enzyme may comprise orthe Super piggyBac™ transposase enzyme may further comprise an aminoacid substitution at positions 103, 194, 372, 375, 450, 509 and 570 ofthe sequence of SEQ ID NO: 4 or SEQ ID NO: 5. In certain embodiments,the amino acid substitution at position 103 of SEQ ID NO: 4 or SEQ IDNO: 5 is a substitution of a proline (P) for a serine (S). In certainembodiments, the amino acid substitution at position 194 of SEQ ID NO: 4or SEQ ID NO: 5 is a substitution of a valine (V) for a methionine (M).In certain embodiments, the amino acid substitution at position 372 ofSEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of an alanine (A) for anarginine (R). In certain embodiments, the amino acid substitution atposition 375 of SEQ ID NO: 4 or SEQ ID NO: 5 is a substitution of analanine (A) for a lysine (K). In certain embodiments, the amino acidsubstitution at position 450 of SEQ ID NO: 4 or SEQ ID NO: 5 is asubstitution of an asparagine (N) for an aspartic acid (D). In certainembodiments, the amino acid substitution at position 509 of SEQ ID NO: 4or SEQ ID NO: 5 is a substitution of a glycine (G) for a serine (S). Incertain embodiments, the amino acid substitution at position 570 of SEQID NO: 4 or SEQ ID NO: 5 is a substitution of a serine (S) for anasparagine (N). In certain embodiments, the piggyBac™ transposase enzymemay comprise a substitution of a valine (V) for a methionine (M) atposition 194 of SEQ ID NO: 4. In certain embodiments, including thoseembodiments wherein the piggyBac™ transposase enzyme may comprise asubstitution of a valine (V) for a methionine (M) at position 194 of SEQID NO: 4, the piggyBac™ transposase enzyme may further comprise an aminoacid substitution at positions 372, 375 and 450 of the sequence of SEQID NO: 4 or SEQ ID NO: 5. In certain embodiments, the piggyBac™transposase enzyme may comprise a substitution of a valine (V) for amethionine (M) at position 194 of SEQ ID NO: 4, a substitution of analanine (A) for an arginine (R) at position 372 of SEQ ID NO: 4, and asubstitution of an alanine (A) for a lysine (K) at position 375 of SEQID NO: 4. In certain embodiments, the piggyBac™ transposase enzyme maycomprise a substitution of a valine (V) for a methionine (M) at position194 of SEQ ID NO: 4, a substitution of an alanine (A) for an arginine(R) at position 372 of SEQ ID NO: 4, a substitution of an alanine (A)for a lysine (K) at position 375 of SEQ ID NO: 4 and a substitution ofan asparagine (N) for an aspartic acid (D) at position 450 of SEQ ID NO:4.

By “introducing” is intended presenting to the plant the polynucleotideconstruct in such a manner that the construct gains access to theinterior of the host cell. The methods of the invention do not depend ona particular method for introducing a polynucleotide construct into ahost cell, only that the polynucleotide construct gains access to theinterior of one cell of the host. Methods for introducing polynucleotideconstructs into bacteria, plants, fungi and animals are known in the artincluding, but not limited to, stable transformation methods, transienttransformation methods, and virus-mediated methods.

As used throughout the disclosure, the term “endogenous” refers tonucleic acid or protein sequence naturally associated with a target geneor a host cell into which it is introduced.

By “stable transformation” is intended that the polynucleotide constructintroduced into a plant integrates into the genome of the host and iscapable of being inherited by progeny thereof.

By “transient transformation” is intended that a polynucleotideconstruct introduced into the host does not integrate into the genome ofthe host.

In preferred embodiments, the piggyBac transposon system is used tointroduce exogenous sequences into a primary human T cell by stabletransformation to generate a modified T_(SCM) or T_(CM).

Additional Transposon Systems

In certain embodiments of the methods of the disclosure, the transposonis a Sleeping Beauty transposon. In certain embodiments, and, inparticular, those embodiments wherein the transposon is a SleepingBeauty transposon, the transposase is a Sleeping Beauty transposase or ahyperactive Sleeping Beauty transposase (SB100X).

The disclosure provides a method of producing a modified stem memoryT-cell (T_(SCM)) or a modified central memory T-cell (T_(CM)),comprising introducing into a primary human T cell (a) a transposoncomposition comprising a transposon comprising an antigen receptor or atherapeutic protein and (b) a transposase composition comprising atransposase or a sequence encoding the transposase; to produce amodified T cell, wherein the modified T cell expresses one or morecell-surface marker(s) of a modified stem memory T-cell (T_(SCM)) or amodified central memory T-cell (T_(CM)), thereby producing a modifiedstem memory T-cell (T_(SCM)) or a modified central memory T-cell(T_(CM)). The disclosure provides a method of producing a plurality ofmodified stem memory T-cells (T_(SCM)) or a plurality of modifiedcentral memory T-cells (T_(CM)), comprising introducing into a pluralityof primary human T cells (a) a transposon composition comprising atransposon comprising an antigen receptor and (b) a transposasecomposition comprising a transposase or a sequence encoding thetransposase; to produce a plurality of modified T cells, wherein atleast 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 65%,70%, 75%, 80%, 85%, 90%, 95%, 99% or any percentage in between of theplurality of modified T cells expresses one or more cell-surfacemarker(s) of a stem memory T-cell (T_(SCM)) or a central memory T-cell(T_(CM)), thereby producing a plurality of modified stem memory T-cells(T_(SCM)) or a plurality of modified central memory T-cells (T_(CM)).

In certain embodiments of the methods of the disclosure, the transposonis a Sleeping Beauty transposon. In certain embodiments, and, inparticular, those embodiments wherein the transposon is a SleepingBeauty transposon, the transposase is a Sleeping Beauty transposase or ahyperactive Sleeping Beauty transposase (SB100X).

In certain embodiments of the methods of the disclosure, the SleepingBeauty transposase enzyme comprises an amino acid sequence at least 75%,80%, 85%, 90%, 95%, 99% or any percentage in between identical to:

(SEQ ID NO: 6)  1 MGKSKEISQD LRKKIVDLHK SGSSLGAISK RLKVPRSSVQ TIVRKYKHHG TTQPSYRSGR 61 RRYLSPRDER TLVRKVQINP RTTAKDLVKM LEETGTKVSI STVKRVLYRH NLKGRSARKK121 PLLQNRHKKA RLRFATAHGD KDRTFWRNVL WSDETKIELF GHNDHRYVWR KKGEACKPKN181 TIPTVKHGGG SIMLWGCFAA GGTGALHKID GIMRKENYVD ILKQHLKTSV RKLKLGRKWV241 FQMDNDPKHT SKVVAKWLKD NKVKVLEWPS QSPDLNPIEN LWAELKKRVR ARRPTNLTQL301 HQLCQEEWAK IHPTYCGKLV EGYPKRLTQV KQFKGNATKY.

In certain embodiments of the methods of the disclosure, the hyperactiveSleeping Beauty (SB100X) transposase enzyme comprises an amino acidsequence at least 75%, 80%, 85%, 90%, 95%, 99% or any percentage inbetween identical to:

(SEQ ID NO: 7)  1 MGKSKEISQD LRKRIVDLHK SGSSLGAISK RLAVPRSSVQ TIVRKYKHHG TTQPSYRSGR 61 RRYLSPRDER TLVRKVQINP RTTAKDLVKM LEETGTKVSI STVKRVLYRH NLKGHSARKK121 PLLQNRHKKA RLRFATAHGD KDRTFWRNVL WSDETKIELF GHNDHRYVWR KKGEACKPKN181 TIPTVKHGGG SIMLWGCFAA GGTGALHKID GIMDAVQYVD ILKQHLKTSV RKLKLGRKWV241 FQHDNDPKHT SKVVAKWLKD NKVKVLEWPS QSPDLNPIEN LWAELKKRVR ARRPTNLTQL301 HQLCQEEWAK IHPNYCGKLV EGYPKRLTQV KQFKGNATKY.

In certain embodiments of the methods of the disclosure, the transposaseis a Helitron transposase. Helitron transposases mobilize the Helraisertransposon, an ancient element from the bat genome that was active about30 to 36 million years ago. An exemplary Helraiser transposon of thedisclosure includes Helibat1, which comprises a nucleic acid sequencecomprising:

(SEQ ID NO: 27)   1 TCCTATATAA TAAAAGAGAA ACATGCAAAT TGACCATCCC TCCGCTACGC TCAAGCCACG  61 CCCACCAGCC AATCAGAAGT GACTATGCAA ATTAACCCAA CAAAGATGGC AGTTAAATTT 121 GCATACGCAG GTGTCAAGCG CCCCAGGAGG CAACGGCGGC CGCGGGCTCC CAGGACCTTC 181 GCTGGCCCCG GGAGGCGAGG CCGGCCGCGC CTAGCCACAC CCGCGGGCTC CCGGGACCTT 241 CGCCAGCAGA GAGCAGAGCG GGAGAGCGGG CGGAGAGCGG GAGGTTTGGA GGACTTGGCA 301 GAGCAGGAGG CCGCTGGACA TAGAGCAGAG CGAGAGAGAG GGTGGCTTGG AGGGCGTGGC 361 TCCCTCTGTC ACCCCAGCTT CCTCATCACA GCTGTGGAAA CTGACAGCAG GGAGGAGGAA 421 GTCCCACCCC CACAGAATCA GCCAGAATCA GCCGTTGGTC AGACAGCTCT CAGCGGCCTG 481 ACAGCCAGGA CTCTCATTCA CCTGCATCTC AGACCGTGAC AGTAGAGAGG TGGGACTATG 541 TCTAAAGAAC AACTGTTGAT ACAACGTAGC TCTGCAGCCG AAAGATGCCG GCGTTATCGA 601 CAGAAAATGT CTGCAGAGCA ACGTGCGTCT GATCTTGAAA GAAGGCGGCG CCTGCAACAG 661 AATGTATCTG AAGAGCAGCT ACTGGAAAAA CGTCGCTCTG AAGCCGAAAA ACAGCGGCGT 721 CATCGACAGA AAATGTCTAA AGACCAACGT GCCTTTGAAG TTGAAAGAAG GCGGTGGCGA 781 CGACAGAATA TGTCTAGAGA ACAGTCATCA ACAAGTACTA CCAATACCGG TAGGAACTGC 841 CTTCTCAGCA AAAATGGAGT ACATGAGGAT GCAATTCTCG AACATAGTTG TGGTGGAATG 901 ACTGTTCGAT GTGAATTTTG CCTATCACTA AATTTCTCTG ATGAAAAACC ATCCGATGGG 961 AAATTTACTC GATGTTGTAG CAAAGGGAAA GTCTGTCCAA ATGATATACA TTTTCCAGAT1021 TACCCGGCAT ATTTAAAAAG ATTAATGACA AACGAAGATT CTGACAGTAA AAATTTCATG1081 GAAAATATTC GTTCCATAAA TAGTTCTTTT GCTTTTGCTT CCATGGGTGC AAATATTGCA1141 TCGCCATCAG GATATGGGCC ATACTGTTTT AGAATACACG GACAAGTTTA TCACCGTACT1201 GGAACTTTAC ATCCTTCGGA TGGTGTTTCT CGGAAGTTTG CTCAACTCTA TATTTTGGAT1261 ACAGCCGAAG CTACAAGTAA AAGATTAGCA ATGCCAGAAA ACCAGGGCTG CTCAGAAAGA1321 CTCATGATCA ACATCAACAA CCTCATGCAT GAAATAAATG AATTAACAAA ATCGTACAAG1381 ATGCTACATG AGGTAGAAAA GGAAGCCCAA TCTGAAGCAG CAGCAAAAGG TATTGCTCCC1441 ACAGAAGTAA CAATGGCGAT TAAATACGAT CGTAACAGTG ACCCAGGTAG ATATAATTCT1501 CCCCGTGTAA CCGAGGTTGC TGTCATATTC AGAAACGAAG ATGGAGAACC TCCTTTTGAA1561 AGGGACTTGC TCATTCATTG TAAACCAGAT CCCAATAATC CAAATGCCAC TAAAATGAAA1621 CAAATCAGTA TCCTGTTTCC TACATTAGAT GCAATGACAT ATCCTATTCT TTTTCCACAT1681 GGTGAAAAAG GCTGGGGAAC AGATATTGCA TTAAGACTCA GAGACAACAG TGTAATCGAC1741 AATAATACTA GACAAAATGT AAGGACACGA GTCACACAAA TGCAGTATTA TGGATTTCAT1801 CTCTCTGTGC GGGACACGTT CAATCCTATT TTAAATGCAG GAAAATTAAC TCAACAGTTT1861 ATTGTGGATT CATATTCAAA AATGGAGGCC AATCGGATAA ATTTCATCAA AGCAAACCAA1921 TCTAAGTTGA GAGTTGAAAA ATATAGTGGT TTGATGGATT ATCTCAAATC TAGATCTGAA1981 AATGACAATG TGCCGATTGG TAAAATGATA ATACTTCCAT CATCTTTTGA GGGTAGTCCC2041 AGAAATATGC AGCAGCGATA TCAGGATGCT ATGGCAATTG TAACGAAGTA TGGCAAGCCC2101 GATTTATTCA TAACCATGAC ATGCAACCCC AAATGGGCAG ATATTACAAA CAATTTACAA2161 CGCTGGCAAA AAGTTGAAAA CAGACCTGAC TTGGTAGCCA GAGTTTTTAA TATTAAGCTG2221 AATGCTCTTT TAAATGATAT ATGTAAATTC CATTTATTTG GCAAAGTAAT AGCTAAAATT2281 CATGTCATTG AATTTCAGAA ACGCGGACTG CCTCACGCTC ACATATTATT GATATTAGAT2341 AGTGAGTCCA AATTACGTTC AGAAGATGAC ATTGACCGTA TAGTTAAGGC AGAAATTCCA2401 GATGAAGACC AGTGTCCTCG ACTTTTTCAA ATTGTAAAAT CAAATATGGT ACATGGACCA2461 TGTGGAATAC AAAATCCAAA TAGTCCATGT ATGGAAAATG GAAAATGTTC AAAGGGATAT2521 CCAAAAGAAT TTCAAAATGC GACCATTGGA AATATTGATG GATATCCCAA ATACAAACGA2581 AGATCTGGTA GCACCATGTC TATTGGAAAT AAAGTTGTCG ATAACACTTG GATTGTCCCT2641 TATAACCCGT ATTTGTGCCT TAAATATAAC TGTCATATAA ATGTTGAAGT CTGTGCATCA2701 ATTAAAAGTG TCAAATATTT ATTTAAATAC ATCTATAAAG GGCACGATTG TGCAAATATT2761 CAAATTTCTG AAAAAAATAT TATCAATCAT GACGAAGTAC AGGACTTCAT TGACTCCAGG2821 TATGTGAGCG CTCCTGAGGC TGTTTGGAGA CTTTTTGCAA TGCGAATGCA TGACCAATCT2881 CATGCAATCA CAAGATTAGC TATTCATTTG CCAAATGATC AGAATTTGTA TTTTCATACC2941 GATGATTTTG CTGAAGTTTT AGATAGGGCT AAAAGGCATA ACTCGACTTT GATGGCTTGG3001 TTCTTATTGA ATAGAGAAGA TTCTGATGCA CGTAATTATT ATTATTGGGA GATTCCACAG3061 CATTATGTGT TTAATAATTC TTTGTGGACA AAACGCCGAA AGGGTGGGAA TAAAGTATTA3121 GGTAGACTGT TCACTGTGAG CTTTAGAGAA CCAGAACGAT ATTACCTTAG ACTTTTGCTT3181 CTGCATGTAA AAGGTGCGAT AAGTTTTGAG GATCTGCGAA CTGTAGGAGG TGTAACTTAT3241 GATACATTTC ATGAAGCTGC TAAACACCGA GGATTATTAC TTGATGACAC TATCTGGAAA3301 GATACGATTG ACGATGCAAT CATCCTTAAT ATGCCCAAAC AACTACGGCA ACTTTTTGCA3361 TATATATGTG TGTTTGGATG TCCTTCTGCT GCAGACAAAT TATGGGATGA GAATAAATCT3421 CATTTTATTG AAGATTTCTG TTGGAAATTA CACCGAAGAG AAGGTGCCTG TGTGAACTGT3481 GAAATGCATG CCCTTAACGA AATTCAGGAG GTATTCACAT TGCATGGAAT GAAATGTTCA3541 CATTTCAAAC TTCCGGACTA TCCTTTATTA ATGAATGCAA ATACATGTGA TCAATTGTAC3601 GAGCAACAAC AGGCAGAGGT TTTGATAAAT TCTCTGAATG ATGAACAGTT GGCAGCCTTT3661 CAGACTATAA CTTCAGCCAT CGAAGATCAA ACTGTACACC CCAAATGCTT TTTCTTGGAT3721 GGTCCAGGTG GTAGTGGAAA AACATATCTG TATAAAGTTT TAACACATTA TATTAGAGGT3781 CGTGGTGGTA CTGTTTTACC CACAGCATCT ACAGGAATTG CTGCAAATTT ACTTCTTGGT3841 GGAAGAACCT TTCATTCCCA ATATAAATTA CCAATTCCAT TAAATGAAAC TTCAATTTCT3901 AGACTCGATA TAAAGAGTGA AGTTGCTAAA ACCATTAAAA AGGCCCAACT TCTCATTATT3961 GATGAATGCA CCATGGCATC CAGTCATGCT ATAAACGCCA TAGATAGATT ACTAAGAGAA4021 ATTATGAATT TGAATGTTGC ATTTGGTGGG AAAGTTCTCC TTCTCGGAGG GGATTTTCGA4081 CAATGTCTCA GTATTGTACC ACATGCTATG CGATCGGCCA TAGTACAAAC GAGTTTAAAG4141 TACTGTAATG TTTGGGGATG TTTCAGAAAG TTGTCTCTTA AAACAAATAT GAGATCAGAG4201 GATTCTGCTT ATAGTGAATG GTTAGTAAAA CTTGGAGATG GCAAACTTGA TAGCAGTTTT4261 CATTTAGGAA TGGATATTAT TGAAATCCCC CATGAAATGA TTTGTAACGG ATCTATTATT4321 GAAGCTACCT TTGGAAATAG TATATCTATA GATAATATTA AAAATATATC TAAACGTGCA4381 ATTCTTTGTC CAAAAAATGA GCATGTTCAA AAATTAAATG AAGAAATTTT GGATATACTT4441 GATGGAGATT TTCACACATA TTTGAGTGAT GATTCCATTG ATTCAACAGA TGATGCTGAA4501 AAGGAAAATT TTCCCATCGA ATTTCTTAAT AGTATTACTC CTTCGGGAAT GCCGTGTCAT4561 AAATTAAAAT TGAAAGTGGG TGCAATCATC ATGCTATTGA GAAATCTTAA TAGTAAATGG4621 GGTCTTTGTA ATGGTACTAG ATTTATTATC AAAAGATTAC GACCTAACAT TATCGAAGCT4681 GAAGTATTAA CAGGATCTGC AGAGGGAGAG GTTGTTCTGA TTCCAAGAAT TGATTTGTCC4741 CCATCTGACA CTGGCCTCCC ATTTAAATTA ATTCGAAGAC AGTTTCCCGT GATGCCAGCA4801 TTTGCGATGA CTATTAATAA ATCACAAGGA CAAACTCTAG ACAGAGTAGG AATATTCCTA4861 CCTGAACCCG TTTTCGCACA TGGTCAGTTA TATGTTGCTT TCTCTCGAGT TCGAAGAGCA4921 TGTGACGTTA AAGTTAAAGT TGTAAATACT TCATCACAAG GGAAATTAGT CAAGCACTCT4981 GAAAGTGTTT TTACTCTTAA TGTGGTATAC AGGGAGATAT TAGAATAAGT TTAATCACTT5041 TATCAGTCAT TGTTTGCATC AATGTTGTTT TTATATCATG TTTTTGTTGT TTTTATATCA5101 TGTCTTTGTT GTTGTTATAT CATGTTGTTA TTGTTTATTT ATTAATAAAT TTATGTATTA5161 TTTTCATATA CATTTTACTC ATTTCCTTTC ATCTCTCACA CTTCTATTAT AGAGAAAGGG5221 CAAATAGCAA TATTAAAATA TTTCCTCTAA TTAATTCCCT TTCAATGTGC ACGAATTTCG5281 TGCACCGGGC CACTAG.

Unlike other transposases, the Helitron transposase does not contain anRNase-H like catalytic domain, but instead comprises a RepHel motif madeup of a replication initiator domain (Rep) and a DNA helicase domain.The Rep domain is a nuclease domain of the HUH superfamily of nucleases.

An exemplary Helitron transposase of the disclosure comprises an aminoacid sequence comprising:

(SEQ ID NO: 28)   1 MSKEQLLIQR SSAAERCRRY RQKMSAEQRA SDLERRRRLQ QNVSEEQLLE KRRSEAEKQR  61 RHRQKMSKDQ RAFEVERRRW RRQNMSREQS STSTTNTGRN CLLSKNGVHE DAILEHSCGG 121 MTVRCEFCLS LNFSDEKPSD GKFTRCCSKG KVCPNDIHFP DYPAYLKRLM TNEDSDSKNF 181 MENIRSINSS FAFASMGANI ASPSGYGPYC FRIHGQVYHR TGTLHPSDGV SRKFAQLYIL 241 DTAEATSKRL AMPENQGCSE RLMININNLM HEINELTKSY KMLHEVEKEA QSEAAAKGIA 301 PTEVTMAIKY DRNSDPGRYN SPRVTEVAVI FRNEDGEPPF ERDLLIHCKP DPNNPNATKM 361 KQISILFPTL DAMTYPILFP HGEKGWGTDI ALRLRDNSVI DNNTRQNVRT RVTQMQYYGF 421 HLSVRDTFNP ILNAGKLTQQ FIVDSYSKME ANRINFIKAN QSKLRVEKYS GLMDYLKSRS 481 ENDNVPIGKM IILPSSFEGS PRNMQQRYQD AMAIVTKYGK PDLFITMTCN PKWADITNNL 541 QRWQKVENRP DLVARVFNIK LNALLNDICK FHLFGKVIAK IHVIEFQKRG LPHAHILLIL 601 DSESKLRSED DIDRIVKAEI PDEDQCPRLF QIVKSNMVHG PCGIQNPNSP CMENGKCSKG 661 YPKEFQNATI GNIDGYPKYK RRSGSTMSIG NKVVDNTWIV PYNPYLCLKY NCHINVEVCA 721 SIKSVKYLFK YIYKGHDCAN IQISEKNIIN HDEVQDFIDS RYVSAPEAVW RLFAMRMHDQ 781 SHAITRLAIH LPNDQNLYFH TDDFAEVLDR AKRHNSTLMA WFLLNREDSD ARNYYYWEIP 841 QHYVFNNSLW TKRRKGGNKV LGRLFTVSFR EPERYYLRLL LLHVKGAISF EDLRTVGGVT 901 YDTFHEAAKH RGLLLDDTIW KDTIDDAIIL NMPKQLRQLF AYICVFGCPS AADKLWDENK 961 SHFIEDFCWK LHRREGACVN CEMHALNEIQ EVFTLHGMKC SHFKLPDYPL LMNANTCDQL1021 YEQQQAEVLI NSLNDEQLAA FQTITSAIED QTVHPKCFFL DGPGGSGKTY LYKVLTHYIR1081 GRGGTVLPTA STGIAANLLL GGRTFHSQYK LPIPLNETSI SRLDIKSEVA KTIKKAQLLI1141 IDECTMASSH AINAIDRLLR EIMNLNVAFG GKVLLLGGDF RQCLSIVPHA MRSAIVQTSL1201 KYCNVWGCFR KLSLKTNMRS EDSAYSEWLV KLGDGKLDSS FHLGMDIIEI PHEMICNGSI1261 IEATFGNSIS IDNIKNISKR AILCPKNEHV QKLNEEILDI LDGDFHTYLS DDSIDSTDDA1321 EKENFPIEFL NSITPSGMPC HKLKLKVGAI IMLLRNLNSK WGLCNGTRFI IKRLRPNIIE1381 AEVLTGSAEG EVVLIPRIDL SPSDTGLPFK LIRRQFPVMP AFAMTINKSQ GQTLDRVGIF1441 LPEPVFAHGQ LYVAFSRVRR ACDVKVKVVN TSSQGKLVKH SESVFTLNVV YREILE.

In Helitron transpositions, a hairpin close to the 3′ end of thetransposon functions as a terminator. However, this hairpin can bebypassed by the transposase, resulting in the transduction of flankingsequences. In addition, Helraiser transposition generates covalentlyclosed circular intermediates. Furthermore, Helitron transpositions canlack target site duplications. In the Helraiser sequence, thetransposase is flanked by left and right terminal sequences termed LTSand RTS. These sequences terminate with a conserved 5′-TC/CTAG-3′ motif.A 19 bp palindromic sequence with the potential to form the hairpintermination structure is located 11 nucleotides upstream of the RTS andconsists of the sequence GTGCACGAATTTCGTGCACCGGGCCACTAG (SEQ ID NO: 29).

In certain embodiments of the methods of the disclosure, the transposaseis a Tol2 transposase. Tol2 transposons may be isolated or derived fromthe genome of the medaka fish, and may be similar to transposons of thehAT family. Exemplary Tol2 transposons of the disclosure are encoded bya sequence comprising about 4.7 kilobases and contain a gene encodingthe Tol2 transposase, which contains four exons. An exemplary Tol2transposase of the disclosure comprises an amino acid sequencecomprising the following:

(SEQ ID NO: 30)  1 MEEVCDSSAA ASSTVQNQPQ DQEHPWPYLR EFFSLSGVNK DSFKMKCVLC LPLNKEISAF 61 KSSPSNLRKH IERMHPNYLK NYSKLTAQKR KIGTSTHASS SKQLKVDSVF PVKHVSPVTV121 NKAILRYIIQ GLHPFSTVDL PSFKELISTL QPGISVITRP TLRSKIAEAA LIMKQKVTAA181 MSEVEWIATT TDCWTARRKS FIGVTAHWIN PGSLERHSAA LACKRLMGSH TFEVLASAMN241 DIHSEYEIRD KVVCTTTDSG SNFMKAFRVF GVENNDIETE ARRCESDDTD SEGCGEGSDG301 VEFQDASRVL DQDDGFEFQL PKHQKCACHL LNLVSSVDAQ KALSNEHYKK LYRSVFGKCQ361 ALWNKSSRSA LAAEAVESES RLQLLRPNQT RWNSTFMAVD RILQICKEAG EGALRNICTS421 LEVPMFNPAE MLFLTEWANT MRPVAKVLDI LQAETNTQLG WLLPSVHQLS LKLQRLHHSL481 RYCDPLVDAL QQGIQTRFKH MFEDPEIIAA AILLPKFRTS WTNDETIIKR GMDYIRVHLE541 PLDHKKELAN SSSDDEDFFA SLKPTTHEAS KELDGYLACV SDTRESLLTF PAICSLSIKT601 NTPLPASAAC ERLFSTAGLL FSPKRARLDT NNFENQLLLK LNLRFYNFE.

An exemplary Tol2 transposon of the disclosure, including invertedrepeats, subterminal sequences and the Tol2 transposase, is encoded by anucleic acid sequence comprising the following:

(SEQ ID NO: 31)   1 CAGAGGTGTA AAGTACTTGA GTAATTTTAC TTGATTACTG TACTTAAGTA TTATTTTTGG  61 GGATTTTTAC TTTACTTGAG TACAATTAAA AATCAATACT TTTACTTTTA CTTAATTACA 121 TTTTTTTAGA AAAAAAAGTA CTTTTTACTC CTTACAATTT TATTTACAGT CAAAAAGTAC 181 TTATTTTTTG GAGATCACTT CATTCTATTT TCCCTTGCTA TTACCAAACC AATTGAATTG 241 CGCTGATGCC CAGTTTAATT TAAATGTTAT TTATTCTGCC TATGAAAATC GTTTTCACAT 301 TATATGAAAT TGGTCAGACA TGTTCATTGG TCCTTTGGAA GTGACGTCAT GTCACATCTA 361 TTACCACAAT GCACAGCACC TTGACCTGGA AATTAGGGAA ATTATAACAG TCAATCAGTG 421 GAAGAAAATG GAGGAAGTAT GTGATTCATC AGCAGCTGCG AGCAGCACAG TCCAAAATCA 481 GCCACAGGAT CAAGAGCACC CGTGGCCGTA TCTTCGCGAA TTCTTTTCTT TAAGTGGTGT 541 AAATAAAGAT TCATTCAAGA TGAAATGTGT CCTCTGTCTC CCGCTTAATA AAGAAATATC 601 GGCCTTCAAA AGTTCGCCAT CAAACCTAAG GAAGCATATT GAGGTAAGTA CATTAAGTAT 661 TTTGTTTTAC TGATAGTTTT TTTTTTTTTT TTTTTTTTTT TTTTTGGGTG TGCATGTTTT 721 GACGTTGATG GCGCGCCTTT TATATGTGTA GTAGGCCTAT TTTCACTAAT GCATGCGATT 781 GACAATATAA GGCTCACGTA ATAAAATGCT AAAATGCATT TGTAATTGGT AACGTTAGGT 841 CCACGGGAAA TTTGGCGCCT ATTGCAGCTT TGAATAATCA TTATCATTCC GTGCTCTCAT 901 TGTGTTTGAA TTCATGCAAA ACACAAGAAA ACCAAGCGAG AAATTTTTTT CCAAACATGT 961 TGTATTGTCA AAACGGTAAC ACTTTACAAT GAGGTTGATT AGTTCATGTA TTAACTAACA1021 TTAAATAACC ATGAGCAATA CATTTGTTAC TGTATCTGTT AATCTTTGTT AACGTTAGTT1081 AATAGAAATA CAGATGTTCA TTGTTTGTTC ATGTTAGTTC ACAGTGCATT AACTAATGTT1141 AACAAGATAT AAAGTATTAG TAAATGTTGA AATTAACATG TATACGTGCA GTTCATTATT1201 AGTTCATGTT AACTAATGTA GTTAACTAAC GAACCTTATT GTAAAAGTGT TACCATCAAA1261 ACTAATGTAA TGAAATCAAT TCACCCTGTC ATGTCAGCCT TACAGTCCTG TGTTTTTGTC1321 AATATAATCA GAAATAAAAT TAATGTTTGA TTGTCACTAA ATGCTACTGT ATTTCTAAAA1381 TCAACAAGTA TTTAACATTA TAAAGTGTGC AATTGGCTGC AAATGTCAGT TTTATTAAAG1441 GGTTAGTTCA CCCAAAAATG AAAATAATGT CATTAATGAC TCGCCCTCAT GTCGTTCCAA1501 GCCCGTAAGA CCTCCGTTCA TCTTCAGAAC ACAGTTTAAG ATATTTTAGA TTTAGTCCGA1561 GAGCTTTCTG TGCCTCCATT GAGAATGTAT GTACGGTATA CTGTCCATGT CCAGAAAGGT1621 AATAAAAACA TCAAAGTAGT CCATGTGACA TCAGTGGGTT AGTTAGAATT TTTTGAAGCA1681 TCGAATACAT TTTGGTCCAA AAATAACAAA ACCTACGACT TTATTCGGCA TTGTATTCTC1741 TTCCGGGTCT GTTGTCAATC CGCGTTCACG ACTTCGCAGT GACGCTACAA TGCTGAATAA1801 AGTCGTAGGT TTTGTTATTT TTGGACCAAA ATGTATTTTC GATGCTTCAA ATAATTCTAC1861 CTAACCCACT GATGTCACAT GGACTACTTT GATGTTTTTA TTACCTTTCT GGACATGGAC1921 AGTATACCGT ACATACATTT TCAGTGGAGG GACAGAAAGC TCTCGGACTA AATCTAAAAT1981 ATCTTAAACT GTGTTCCGAA GATGAACGGA GGTGTTACGG GCTTGGAACG ACATGAGGGT2041 GAGTCATTAA TGACATCTTT TCATTTTTGG GTGAACTAAC CCTTTAATGC TGTAATCAGA2101 GAGTGTATGT GTAATTGTTA CATTTATTGC ATACAATATA AATATTTATT TGTTGTTTTT2161 ACAGAGAATG CACCCAAATT ACCTCAAAAA CTACTCTAAA TTGACAGCAC AGAAGAGAAA2221 GATCGGGACC TCCACCCATG CTTCCAGCAG TAAGCAACTG AAAGTTGACT CAGTTTTCCC2281 AGTCAAACAT GTGTCTCCAG TCACTGTGAA CAAAGCTATA TTAAGGTACA TCATTCAAGG2341 ACTTCATCCT TTCAGCACTG TTGATCTGCC ATCATTTAAA GAGCTGATTA GTACACTGCA2401 GCCTGGCATT TCTGTCATTA CAAGGCCTAC TTTACGCTCC AAGATAGCTG AAGCTGCTCT2461 GATCATGAAA CAGAAAGTGA CTGCTGCCAT GAGTGAAGTT GAATGGATTG CAACCACAAC2521 GGATTGTTGG ACTGCACGTA GAAAGTCATT CATTGGTGTA ACTGCTCACT GGATCAACCC2581 TGGAAGTCTT GAAAGACATT CCGCTGCACT TGCCTGCAAA AGATTAATGG GCTCTCATAC2641 TTTTGAGGTA CTGGCCAGTG CCATGAATGA TATCCACTCA GAGTATGAAA TACGTGACAA2701 GGTTGTTTGC ACAACCACAG ACAGTGGTTC CAACTTTATG AAGGCTTTCA GAGTTTTTGG2761 TGTGGAAAAC AATGATATCG AGACTGAGGC AAGAAGGTGT GAAAGTGATG ACACTGATTC2821 TGAAGGCTGT GGTGAGGGAA GTGATGGTGT GGAATTCCAA GATGCCTCAC GAGTCCTGGA2881 CCAAGACGAT GGCTTCGAAT TCCAGCTACC AAAACATCAA AAGTGTGCCT GTCACTTACT2941 TAACCTAGTC TCAAGCGTTG ATGCCCAAAA AGCTCTCTCA AATGAACACT ACAAGAAACT3001 CTACAGATCT GTCTTTGGCA AATGCCAAGC TTTATGGAAT AAAAGCAGCC GATCGGCTCT3061 AGCAGCTGAA GCTGTTGAAT CAGAAAGCCG GCTTCAGCTT TTAAGGCCAA ACCAAACGCG3121 GTGGAATTCA ACTTTTATGG CTGTTGACAG AATTCTTCAA ATTTGCAAAG AAGCAGGAGA3181 AGGCGCACTT CGGAATATAT GCACCTCTCT TGAGGTTCCA ATGTAAGTGT TTTTCCCCTC3241 TATCGATGTA AACAAATGTG GGTTGTTTTT GTTTAATACT CTTTGATTAT GCTGATTTCT3301 CCTGTAGGTT TAATCCAGCA GAAATGCTGT TCTTGACAGA GTGGGCCAAC ACAATGCGTC3361 CAGTTGCAAA AGTACTCGAC ATCTTGCAAG CGGAAACGAA TACACAGCTG GGGTGGCTGC3421 TGCCTAGTGT CCATCAGTTA AGCTTGAAAC TTCAGCGACT CCACCATTCT CTCAGGTACT3481 GTGACCCACT TGTGGATGCC CTACAACAAG GAATCCAAAC ACGATTCAAG CATATGTTTG3541 AAGATCCTGA GATCATAGCA GCTGCCATCC TTCTCCCTAA ATTTCGGACC TCTTGGACAA3601 ATGATGAAAC CATCATAAAA CGAGGTAAAT GAATGCAAGC AACATACACT TGACGAATTC3661 TAATCTGGGC AACCTTTGAG CCATACCAAA ATTATTCTTT TATTTATTTA TTTTTGCACT3721 TTTTAGGAAT GTTATATCCC ATCTTTGGCT GTGATCTCAA TATGAATATT GATGTAAAGT3781 ATTCTTGCAG CAGGTTGTAG TTATCCCTCA GTGTTTCTTG AAACCAAACT CATATGTATC3841 ATATGTGGTT TGGAAATGCA GTTAGATTTT ATGCTAAAAT AAGGGATTTG CATGATTTTA3901 GATGTAGATG ACTGCACGTA AATGTAGTTA ATGACAAAAT CCATAAAATT TGTTCCCAGT3961 CAGAAGCCCC TCAACCAAAC TTTTCTTTGT GTCTGCTCAC TGTGCTTGTA GGCATGGACT4021 ACATCAGAGT GCATCTGGAG CCTTTGGACC ACAAGAAGGA ATTGGCCAAC AGTTCATCTG4081 ATGATGAAGA TTTTTTCGCT TCTTTGAAAC CGACAACACA TGAAGCCAGC AAAGAGTTGG4141 ATGGATATCT GGCCTGTGTT TCAGACACCA GGGAGTCTCT GCTCACGTTT CCTGCTATTT4201 GCAGCCTCTC TATCAAGACT AATACACCTC TTCCCGCATC GGCTGCCTGT GAGAGGCTTT4261 TCAGCACTGC AGGATTGCTT TTCAGCCCCA AAAGAGCTAG GCTTGACACT AACAATTTTG4321 AGAATCAGCT TCTACTGAAG TTAAATCTGA GGTTTTACAA CTTTGAGTAG CGTGTACTGG4381 CATTAGATTG TCTGTCTTAT AGTTTGATAA TTAAATACAA ACAGTTCTAA AGCAGGATAA4441 AACCTTGTAT GCATTTCATT TAATGTTTTT TGAGATTAAA AGCTTAAACA AGAATCTCTA4501 GTTTTCTTTC TTGCTTTTAC TTTTACTTCC TTAATACTCA AGTACAATTT TAATGGAGTA4561 CTTTTTTACT TTTACTCAAG TAAGATTCTA GCCAGATACT TTTACTTTTA ATTGAGTAAA4621 ATTTTCCCTA AGTACTTGTA CTTTCACTTG AGTAAAATTT TTGAGTACTT TTTACACCTC4681 TG.

Homologous Recombination

In certain embodiments of the methods of the disclosure, a modifiedCAR-T_(SCM) or CAR-T_(CM) of the disclosure is produced by introducingan antigen receptor into a primary human T cell of the disclosure byhomologous recombination. In certain embodiments of the disclosure, thehomologous recombination is induced by a single or double strand breakinduced by a genomic editing composition or construct of the disclosure.Homologous recombination methods of the disclosure comprise contacting agenomic editing composition or construct of the disclosure to a genomicsequence to induce at least one break in the sequence and to provide anentry point in the genomic sequence for an exogenous donor sequencecomposition. Donor sequence compositions of the disclosure areintegrated into the genomic sequence at the induced entry point by thecell's native DNA repair machinery.

In certain embodiments of the methods of the disclosure, homologousrecombination introduces a sequence encoding an antigen receptor and/ora donor sequence composition of the disclosure into a “genomic safeharbor” site. In certain embodiments, a mammalian genomic sequencecomprises the genomic safe harbor site. In certain embodiments, aprimate genomic sequence comprises the genomic safe harbor site. Incertain embodiments, a human genomic sequence comprises the genomic safeharbor site.

Genomic safe harbor sites are able to accommodate the integration of newgenetic material in a manner that ensures that the newly insertedgenetic elements function reliably (for example, are expressed at atherapeutically effective level of expression) and do not causedeleterious alterations to the host genome that cause a risk to the hostorganism. Potential genomic safe harbors include, but are not limitedto, intronic sequences of the human albumin gene, the adeno-associatedvirus site 1 (AAVS1), a naturally occurring site of integration of AAVvirus on chromosome 19, the site of the chemokine (C-C motif) receptor 5(CCR5) gene and the site of the human ortholog of the mouse Rosa26locus.

In certain embodiments of the methods of the disclosure, homologousrecombination introduces a sequence encoding an antigen receptor and/ora donor sequence composition of the disclosure into a sequence encodingone or more components of an endogenous T-cell receptor or a majorhistocompatibility complex (MHC). In certain embodiments, inducinghomologous recombination within a genomic sequence encoding theendogenous T-cell receptor or the MHC disrupts the endogenous gene, andoptionally, replaces part of the coding sequence of the endogenous genewith a donor sequence composition of the disclosure. In certainembodiments, inducing homologous recombination within a genomic sequenceencoding the endogenous T-cell receptor or the MHC disrupts theendogenous gene, and optionally, replaces the entire coding sequence ofthe endogenous gene with a donor sequence composition of the disclosure.In certain embodiments of the methods of the disclosure, introduction ofa sequence encoding an antigen receptor or a donor sequence compositionof the disclosure by homologous recombination operably links the antigenreceptor to an endogenous T cell promoter. In certain embodiments of themethods of the disclosure, introduction of a sequence encoding anantigen receptor or a donor sequence composition of the disclosure byhomologous recombination operably links the antigen receptor or thetherapeutic protein to a transcriptional or translational regulatoryelement. In certain embodiments of the methods of the disclosure,introduction of a sequence encoding an antigen receptor or a donorsequence composition of the disclosure by homologous recombinationoperably links the antigen receptor or the therapeutic protein to atranscriptional regulatory element. In certain embodiments, thetranscriptional regulatory element comprises an endogenous T cell 5′UTR.

In certain embodiments of the introduction step comprising a homologousrecombination, a genomic editing composition contacts a genomic sequenceof at least one primary T cell of the plurality of T cells. In certainembodiments of the introduction step comprising a homologousrecombination, a genomic editing composition contacts a genomic sequenceof a portion of primary T cells of the plurality of T cells. In certainembodiments, the portion of primary T cells is at least 1%, 2%, 5%, 7%,10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, 95%, 97%, 99% or any percentage in between of the totalnumber of primary T cells in the plurality of T cells. In certainembodiments of the introduction step comprising a homologousrecombination, a genomic editing composition contacts a genomic sequenceof each primary T cell of the plurality of T cells. In certainembodiments of the introduction step comprising a homologousrecombination, a genomic editing composition induces a single strandbreak. In certain embodiments of the introduction step comprising ahomologous recombination, a genomic editing composition induces a doublestrand break. In certain embodiments of the introduction step comprisinga homologous recombination, the introduction step further comprises adonor sequence composition. In certain embodiments, the donor sequencecomposition comprises a sequence encoding the antigen receptor. Incertain embodiments, the donor sequence composition comprises a sequenceencoding the antigen receptor, a 5′ genomic sequence and a 3′ genomicsequence, wherein the 5′ genomic sequence is homologous or identical toa genomic sequence of the primary T cell that is 5′ to the break pointinduced by the genomic editing composition and the 3′ genomic sequenceis homologous or identical to a genomic sequence of the primary T cellthat is 3′ to the break point induced by the genomic editingcomposition. In certain embodiments, the 5′ genomic sequence and/or the3′ genomic sequence comprises at least 50 bp, 100 bp, at least 200 bp,at least 300 bp, at least 400 bp, at least 500 bp, at least 600 bp, atleast 700 bp, at least 800 bp, at least 900 bp, at least 1000 bp, atleast 1100 bp, at least 1200 bp, at least 1300 bp, at least 1400, or atleast 1500 bp, at least 1600 bp, at least 1700 bp, at least 1800 bp, atleast 1900 bp, at least 2000 bp in length or any length of base pairs(bp) in between, inclusive of the end points. In certain embodiments ofthe introduction step comprising a homologous recombination, the genomicediting composition and donor sequence composition are contacted withthe genomic sequence simultaneously or sequentially. In certainembodiments of the introduction step comprising a homologousrecombination, the genomic editing composition and donor sequencecomposition are contacted with the genomic sequence sequentially, andthe genomic editing composition is provided first. In certainembodiments of the introduction step comprising a homologousrecombination, the genomic editing composition comprises a sequenceencoding a DNA binding domain and a sequence encoding a nuclease domain.In certain embodiments of the introduction step comprising a homologousrecombination, the genomic editing composition comprises a DNA bindingdomain and a nuclease domain. In certain embodiments of the genomicediting composition, the DNA binding domain comprises a guide RNA(gRNA). In certain embodiments of the genomic editing composition, theDNA binding domain comprises a DNA-binding domain of a TALEN. In certainembodiments of the genomic editing composition, the DNA binding domaincomprises a DNA-binding domain of a ZFN. In certain embodiments of thegenomic editing composition, the nuclease domain comprises a Cas9nuclease or a sequence thereof. In certain embodiments of the genomicediting composition, the nuclease domain comprises an inactive Cas9 (SEQID NO: 33, comprising a substitution of a Alanine (A) for Aspartic Acid(D) at position 10 (D10A) and a substitution of Alanine (A) forHistidine (H) at position 840 (H840A)). In certain embodiments of thegenomic editing composition, the nuclease domain comprises a short andinactive Cas9 (SEQ ID NO: 32, comprising a substitution of an Alanine(A) for an Aspartic Acid (D) at position 10 (D10A) and a substitution ofan Alanine (A) for an Asparagine (N) at position 540 (N540A)). Incertain embodiments of the genomic editing composition, the nucleasedomain comprises or further comprises a type IIS endonuclease. Incertain embodiments of the genomic editing composition, the type IISendonuclease comprises AciI, Mn1I, AlwI, BbvI, BccI, BceAI, BsmAI,BsmFI, BspCNI, BsrI, BtsCI, HgaI, HphI, HpyAV, Mbo1I, My1I, PleI, SfaNI,AcuI, BciVI, BfuAI, BmgBI, BmrI, BpmI, BpuEI, BsaI, BseRI, BsgI, BsmI,BspMI, BsrBI, BsrBI, BsrDI, BtgZI, BtsI, EarI, EciI, MmeI, NmeAIII,BbvCI, Bpu10I, BspQI, SapI, BaeI, BsaXI, CspCI, BfiI, MboII, Acc36I,FokI or Clo051. In certain embodiments, the type IIS endonucleasecomprises Clo051. In certain embodiments of the genomic editingcomposition, the nuclease domain comprises or further comprises a TALENor a nuclease domain thereof. In certain embodiments of the genomicediting composition, the nuclease domain comprises or further comprisesa ZFN or a nuclease domain thereof. In certain embodiments of theintroduction step comprising a homologous recombination, the genomicediting composition induces a break in a genomic sequence and the donorsequence composition is inserted using the endogenous DNA repairmechanisms of the primary T cell. In certain embodiments of theintroduction step comprising a homologous recombination, the insertionof the donor sequence composition eliminates a DNA binding site of thegenomic editing composition, thereby preventing further activity of thegenomic editing composition.

In certain embodiments of the methods of homologous recombination of thedisclosure, the nuclease domain of a genomic editing composition orconstruct is capable of introducing a break at a defined location in agenomic sequence of the primary human T cell, and, furthermore, maycomprise, consist essentially of or consist of, a homodimer or aheterodimer. In certain embodiments, the nuclease is an endonuclease.Effector molecules, including those effector molecules comprising ahomodimer or a heterodimer, may comprise, consist essentially of orconsist of, a Cas9, a Cas9 nuclease domain or a fragment thereof. Incertain embodiments, the Cas9 is a catalytically inactive or“inactivated” Cas9 (dCas9). In certain embodiments, the Cas9 is acatalytically inactive or “inactivated” nuclease domain of Cas9. Incertain embodiments, the dCas9 is encoded by a shorter sequence that isderived from a full length, catalytically inactivated, Cas9, referred toherein as a “small” dCas9 or dSaCas9.

In certain embodiments, the inactivated, small, Cas9 (dSaCas9)operatively-linked to an active nuclease. In certain embodiments, thedisclosure provides a fusion protein comprising, consisting essentiallyof or consisting of a DNA binding domain and molecule nuclease, whereinthe nuclease comprises a small, inactivated Cas9 (dSaCas9). In certainembodiments, the dSaCas9 of the disclosure comprises the mutations D10Aand N580A (underlined and bolded) which inactivate the catalytic site.In certain embodiments, the dSaCas9 of the disclosure comprises theamino acid sequence of:

(SEQ ID NO: 32)    1 MKRNYILGL A IGITSVGYGI IDYETRDVID AGVRLFKEAN VENNEGRRSK RGARRLKRRR  61 RHRIQRVKKL LFDYNLLTDH SELSGINPYE ARVKGLSQKL SEEEFSAALL HLAKRRGVHN 121 VNEVEEDTGN ELSTKEQISR NSKALEEKYV AELQLERLKK DGEVRGSINR FKTSDYVKEA 181 KQLLKVQKAY HQLDQSFIDT YIDLLETRRT YYEGPGEGSP FGWKDIKEWY EMLMGHCTYF 241 PEELRSVKYA YNADLYNALN DLNNLVITRD ENEKLEYYEK FQIIENVFKQ KKKPTLKQIA 301 KEILVNEEDI KGYRVTSTGK PEFTNLKVYH DIKDITARKE IIENAELLDQ IAKILTIYQS 361 SEDIQEELTN LNSELTQEEI EQISNLKGYT GTHNLSLKAI NLILDELWHT NDNQIAIFNR 421 LKLVPKKVDL SQQKEIPTTL VDDFILSPVV KRSFIQSIKV INAIIKKYGL PNDIIIELAR 481 EKNSKDAQKM INEMQKRNRQ TNERIEEIIR TTGKENAKYL IEKIKLHDMQ EGKCLYSLEA 541 IPLEDLLNNP FNYEVDHIIP RSVSFDNSFN NKVLVKQEE A  SKKGNRTPFQ YLSSSDSKIS 601 YETFKKHILN LAKGKGRISK TKKEYLLEER DINRFSVQKD FINRNLVDTR YATRGLMNLL 661 RSYFRVNNLD VKVKSINGGF TSFLRRKWKF KKERNKGYKH HAEDALIIAN ADFIFKEWKK 721 LDKAKKVMEN QMFEEKQAES MPEIETEQEY KEIFITPHQI KHIKDFKDYK YSHRVDKKPN 781 RELINDTLYS TRKDDKGNTL IVNNLNGLYD KDNDKLKKLI NKSPEKLLMY HHDPQTYQKL 841 KLIMEQYGDE KNPLYKYYEE TGNYLTKYSK KDNGPVIKKI KYYGNKLNAH LDITDDYPNS 901 RNKVVKLSLK PYRFDVYLDN GVYKFVTVKN LDVIKKENYY EVNSKCYEEA KKLKKISNQA 961 EFIASFYNND LIKINGELYR VIGVNNDLLN RIEVNMIDIT YREYLENMND KRPPRIIKTI1021 ASKTQSIKKY STDILGNLYE VKSKKHPQII KKG.

In certain embodiments, the dCas9 of the disclosure comprises a dCas9isolated or derived from Staphyloccocus pyogenes. In certainembodiments, the dCas9 comprises a dCas9 with substitutions at positions10 and 840 of the amino acid sequence of the dCas9 which inactivate thecatalytic site. In certain embodiments, these substitutions are D10A andH840A. In certain embodiments, the amino acid sequence of the dCas9comprises the sequence of:

(SEQ ID NO: 33)    1 XDKKYSIGL A IGTNSVGWAV ITDEYKVPSK KFKVLGNTDR HSIKKNLIGA LLFDSGETAE  61 ATRLKRTARR RYTRRKNRIC YLQEIFSNEM AKVDDSFFHR LEESFLVEED KKHERHPIFG 121 NIVDEVAYHE KYPTIYHLRK KLVDSTDKAD LRLIYLALAH MIKFRGHFLI EGDLNPDNSD 181 VDKLFIQLVQ TYNQLFEENP INASGVDAKA ILSARLSKSR RLENLIAQLP GEKKNGLFGN 241 LIALSLGLTP NFKSNFDLAE DAKLQLSKDT YDDDLDNLLA QIGDQYADLF LAAKNLSDAI 301 LLSDILRVNT EITKAPLSAS MIKRYDEHHQ DLTLLKALVR QQLPEKYKEI FFDQSKNGYA 361 GYIDGGASQE EFYKFIKPIL EKMDGTEELL VKLNREDLLR KQRTFDNGSI PHQIHLGELH 421 AILRRQEDFY PFLKDNREKI EKILTFRIPY YVGPLARGNS RFAWMTRKSE ETITPWNFEE 481 VVDKGASAQS FIERMTNFDK NLPNEKVLPK HSLLYEYFTV YNELTKVKYV TEGMRKPAFL 541 SGEQKKAIVD LLFKTNRKVT VKQLKEDYFK KIECFDSVEI SGVEDRFNAS LGTYHDLLKI 601 IKDKDFLDNE ENEDILEDIV LTLTLFEDRE MIEERLKTYA HLFDDKVMKQ LKRRRYTGWG 661 RLSRKLINGI RDKQSGKTIL DFLKSDGFAN RNFMQLIHDD SLTFKEDIQK AQVSGQGDSL 721 HEHIANLAGS PAIKKGILQT VKVVDELVKV MGRHKPENIV IEMARENQTT QKGQKNSRER 781 MKRIEEGIKE LGSQILKEHP VENTQLQNEK LYLYYLQNGR DMYVDQELDI NRLSDYDVD A 841 IVPQSFLKDD SIDNKVLTRS DKNRGKSDNV PSEEVVKKMK NYWRQLLNAK LITQRKFDNL 901 TKAERGGLSE LDKAGFIKRQ LVETRQITKH VAQILDSRMN TKYDENDKLI REVKVITLKS 961 KLVSDFRKDF QFYKVREINN YHHAHDAYLN AVVGTALIKK YPKLESEFVY GDYKVYDVRK1021 MIAKSEQEIG KATAKYFFYS NIMNFFKTEI TLANGEIRKR PLIETNGETG EIVWDKGRDF1081 ATVRKVLSMP QVNIVKKTEV QTGGFSKESI LPKRNSDKLI ARKKDWDPKK YGGFDSPTVA1141 YSVLVVAKVE KGKSKKLKSV KELLGITIME RSSFEKNPID FLEAKGYKEV KKDLIIKLPK1201 YSLFELENGR KRMLASAGEL QKGNELALPS KYVNFLYLAS HYEKLKGSPE DNEQKQLFVE1261 QHKHYLDEII EQISEFSKRV ILADANLDKV LSAYNKHRDK PIREQAENII HLFTLTNLGA1321 PAAFKYFDTT IDRKRYTSTK EVLDATLIHQ SITGLYETRI DLSQLGGD.

In certain embodiments of the disclosure, the nuclease domain maycomprise, consist essentially of or consist of a dCas9 or a dSaCas9 anda type IIS endonuclease. In certain embodiments of the disclosure, thenuclease domain may comprise, consist essentially of or consist of adSaCas9 and a type IIS endonuclease, including, but not limited to,AciI, Mn1I, AlwI, BbvI, BccI, BceAI, BsmAI, BsmFI, BspCNI, BsrI, BtsCI,HgaI, HphI, HpyAV, Mbo1I, My1I, PleI, SfaNI, AcuI, BciVI, BfuAI, BmgBI,BmrI, BpmI, BpuEI, BsaI, BseRI, BsgI, BsmI, BspMI, BsrBI, BsrBI, BsrDI,BtgZI, BtsI, EarI, EciI, MmeI, NmeAIII, BbvCI, Bpu10I, BspQI, SapI,BaeI, BsaXI, CspCI, BfiI, MboII, Acc36I, FokI or Clo051. In certainembodiments of the disclosure, the nuclease domain may comprise, consistessentially of or consist of a dSaCas9 and Clo051. An exemplary Clo051nuclease domain may comprise, consist essentially of or consist of, theamino acid sequence of: EGIKSNISLLKDELRGQISHISHEYLSLIDLAFDSKQNRLFEMKVLELLVNEYGFKGRHLGGSRKPDGIVYSTTLEDNFGIIVDTKAYSEGYSLPISQADEMERYVRENSNRDEEVNPNKWWENFSEEVKKYYFVFISGSFKGKFEEQLRRLSMTTGVNGSAVNVVNLLLGAEKIRSGEMTIEELERAMFNNSEFILKY (SEQ ID NO: 34).

An exemplary dCas9-Clo051 nuclease domain may comprise, consistessentially of or consist of, the amino acid sequence of (Clo051sequence underlined, linker bold italics, dCas9 sequence in italics):

(SEQ ID NO: 40)MAPKKKRKVEGIKSNISLLKDELRGQISHISHEYLSLIDLAFDSKQNRLFEMKVLELLVNEYGFKGRHLGGSRKPDGIVYSTTLEDNFGIIVDTKAYSEGYSLPISQADEMERYVRENSNRDEEVNPNKWWENFSEEVKKYYFVFISGSFKGKFEEQLRRLSMTTGVNGSAVNVVNLLLGAEKIRSGEMTIEELERAMFNNSEFILKY

DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKEDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDGSPKKKRKVSS.

In certain embodiments, the nuclease capable of introducing a break at adefined location in the genomic DNA of the primary human T cell maycomprise, consist essentially of or consist of, a homodimer or aheterodimer. Nuclease domains of the genomic editing compositions orconstructs of the disclosure may comprise, consist essentially of orconsist of a nuclease domain isolated, derived or recombined from atranscription-activator-like effector nuclease (TALEN). TALENs aretranscription factors with programmable DNA binding domains that providea means to create designer proteins that bind to pre-determined DNAsequences or individual nucleic acids. Modular DNA binding domains havebeen identified in transcriptional activator-like (TAL) proteins, or,more specifically, transcriptional activator-like effector nucleases(TALENs), thereby allowing for the de novo creation of synthetictranscription factors that bind to DNA sequences of interest and, ifdesirable, also allowing a second domain present on the protein orpolypeptide to perform an activity related to DNA. TAL proteins havebeen derived from the organisms Xanthomonas and Ralstonia.

In certain embodiments of the disclosure, the nuclease domain of thegenomic editing composition or construct may comprise, consistessentially of or consist of a nuclease domain isolated, derived orrecombined from a TALEN and a type IIS endonuclease. In certainembodiments of the disclosure, the type IIS endonuclease may comprise,consist essentially of or consist of AciI, Mn1I, AlwI, BbvI, BccI,BceAI, BsmAI, BsmFI, BspCNI, BsrI, BtsCI, HgaI, HphI, HpyAV, Mbo1I,My1I, PleI, SfaNI, AcuI, BciVI, BfuAI, BmgBI, BmrI, BpmI, BpuEI, BsaI,BseRI, BsgI, BsmI, BspMI, BsrBI, BsrBI, BsrDI, BtgZI, BtsI, EarI, EciI,MmeI, NmeAIII, BbvCI, Bpu10I, BspQI, SapI, BaeI, BsaXI, CspCI, BfiI,MboII, Acc36I, FokI or Clo051. In certain embodiments of the disclosure,the type IIS endonuclease may comprise, consist essentially of orconsist of Clo051 (SEQ ID NO: 34).

In certain embodiments of the disclosure, the nuclease domain of thegenomic editing composition or construct may comprise, consistessentially of or consist of a nuclease domain isolated, derived orrecombined from a zinc finger nuclease (ZFN) and a type IISendonuclease. In certain embodiments of the disclosure, the type IISendonuclease may comprise, consist essentially of or consist of AciI,Mn1I, AlwI, BbvI, BccI, BceAI, BsmAI, BsmFI, BspCNI, BsrI, BtsCI, HgaI,HphI, HpyAV, Mbo1I, My1I, PleI, SfaNI, AcuI, BciVI, BfuAI, BmgBI, BmrI,BpmI, BpuEI, BsaI, BseRI, BsgI, BsmI, BspMI, BsrBI, BsrBI, BsrDI, BtgZI,BtsI, EarI, EciI, MmeI, NmeAIII, BbvCI, Bpu10I, BspQI, SapI, BaeI,BsaXI, CspCI, BfiI, MboII, Acc36I, FokI or Clo051. In certainembodiments of the disclosure, the type IIS endonuclease may comprise,consist essentially of or consist of Clo051 (SEQ ID NO: 34).

In certain embodiments of the genomic editing compositions or constructsof the disclosure, the DNA binding domain and the nuclease domain may becovalently linked. For example, a fusion protein may comprise the DNAbinding domain and the nuclease domain. In certain embodiments of thegenomic editing compositions or constructs of the disclosure, the DNAbinding domain and the nuclease domain may be operably linked through anon-covalent linkage.

Secreted Proteins from Modified T Cells

In certain embodiments of the composition and methods of the disclosure,modified T-cells express therapeutic proteins. Therapeutic proteins ofthe disclosure include secreted proteins. Preferably, in a therapeuticcontext, the therapeutic protein is a human protein, including asecreted human protein. When expressed or secreted by CAR-T cells of thedisclosure, the combination comprising the CAR-T cell and thetherapeutic protein secreted therefrom may be considered a monotherapy.However, the CAR-T cells of the disclosure may be administered as acombination therapy with a second agent. A database of human secretedproteins that may be expressed or secreted by modified T-cell of thedisclosure can be found atproteinatlas.org/search/protein_class:Predicted %20secreted %20proteins,the contents of which are incorporated herein by reference. Exemplaryhuman secreted proteins are provided, but are not limited to the humansecreted proteins, in Table 1.

TABLE 1 Exemplary Human Secreted Proteins Gene Ensembl ID Genedescription A1BG ENSG00000121410 Alpha-1-B glycoprotein A2MENSG00000175899 Alpha-2-macroglobulin A2ML1 ENSG00000166535Alpha-2-macroglobulin-like 1 A4GNT ENSG00000118017Alpha-1,4-N-acetylglucosaminyltransferase AADACL2 ENSG00000197953Arylacetamide deacetylase-like 2 AANAT ENSG00000129673 AralkylamineN-acetyltransferase ABCG1 ENSG00000160179 ATP-binding cassette,sub-family G (WHITE), member 1 ABHD1 ENSG00000143994 Abhydrolase domaincontaining 1 ABHD10 ENSG00000144827 Abhydrolase domain containing 10ABHD14A ENSG00000248487 Abhydrolase domain containing 14A ABHD15ENSG00000168792 Abhydrolase domain containing 15 ABI3BP ENSG00000154175ABI family, member 3 (NESH) binding protein AC008641.1 ENSG00000279109AC009133.22 ENSG00000277669 AC009491.2 ENSG00000279664 AC011513.3ENSG00000267881 AC136352.5 ENSG00000277666 AC145212.4 ENSG00000277400MaFF-interacting protein AC233755.1 ENSG00000275063 ACACBENSG00000076555 Acetyl-CoA carboxylase beta ACAN ENSG00000157766Aggrecan ACE ENSG00000159640 Angiotensin I converting enzyme ACHEENSG00000087085 Acetylcholinesterase (Yt blood group) ACP2ENSG00000134575 Acid phosphatase 2, lysosomal ACP5 ENSG00000102575 Acidphosphatase 5, tartrate resistant ACP6 ENSG00000162836 Acid phosphatase6, lysophosphatidic ACPP ENSG00000014257 Acid phosphatase, prostate ACRENSG00000100312 Acrosin ACRBP ENSG00000111644 Acrosin binding proteinACRV1 ENSG00000134940 Acrosomal vesicle protein 1 ACSF2 ENSG00000167107Acyl-CoA synthetase family member 2 ACTL10 ENSG00000182584 Actin-like 10ACVR1 ENSG00000115170 Activin A receptor, type I ACVR1C ENSG00000123612Activin A receptor, type IC ACVRL1 ENSG00000139567 Activin A receptortype II-like 1 ACYP1 ENSG00000119640 Acylphosphatase 1, erythrocyte(common) type ACYP2 ENSG00000170634 Acylphosphatase 2, muscle typeADAM10 ENSG00000137845 ADAM metallopeptidase domain 10 ADAM12ENSG00000148848 ADAM metallopeptidase domain 12 ADAM15 ENSG00000143537ADAM metallopeptidase domain 15 ADAM17 ENSG00000151694 ADAMmetallopeptidase domain 17 ADAM18 ENSG00000168619 ADAM metallopeptidasedomain 18 ADAM22 ENSG00000008277 ADAM metallopeptidase domain 22 ADAM28ENSG00000042980 ADAM metallopeptidase domain 28 ADAM29 ENSG00000168594ADAM metallopeptidase domain 29 ADAM32 ENSG00000197140 ADAMmetallopeptidase domain 32 ADAM33 ENSG00000149451 ADAM metallopeptidasedomain 33 ADAM7 ENSG00000069206 ADAM metallopeptidase domain 7 ADAM8ENSG00000151651 ADAM metallopeptidase domain 8 ADAM9 ENSG00000168615ADAM metallopeptidase domain 9 ADAMDEC1 ENSG00000134028 ADAM-like,decysin 1 ADAMTS1 ENSG00000154734 ADAM metallopeptidase withthrombospondin type 1 motif, 1 ADAMTS10 ENSG00000142303 ADAMmetallopeptidase with thrombospondin type 1 motif, 10 ADAMTS12ENSG00000151388 ADAM metallopeptidase with thrombospondin type 1 motif,12 ADAMTS13 ENSG00000160323 ADAM metallopeptidase with thrombospondintype 1 motif, 13 ADAMTS14 ENSG00000138316 ADAM metallopeptidase withthrombospondin type 1 motif, 14 ADAMTS15 ENSG00000166106 ADAMmetallopeptidase with thrombospondin type 1 motif, 15 ADAMTS16ENSG00000145536 ADAM metallopeptidase with thrombospondin type 1 motif,16 ADAMTS17 ENSG00000140470 ADAM metallopeptidase with thrombospondintype 1 motif, 17 ADAMTS18 ENSG00000140873 ADAM metallopeptidase withthrombospondin type 1 motif, 18 ADAMTS19 ENSG00000145808 ADAMmetallopeptidase with thrombospondin type 1 motif, 19 ADAMTS2ENSG00000087116 ADAM metallopeptidase with thrombospondin type 1 motif,2 ADAMTS20 ENSG00000173157 ADAM metallopeptidase with thrombospondintype 1 motif, 20 ADAMTS3 ENSG00000156140 ADAM metallopeptidase withthrombospondin type 1 motif, 3 ADAMTS5 ENSG00000154736 ADAMmetallopeptidase with thrombospondin type 1 motif, 5 ADAMTS6ENSG00000049192 ADAM metallopeptidase with thrombospondin type 1 motif,6 ADAMTS7 ENSG00000136378 ADAM metallopeptidase with thrombospondin type1 motif, 7 ADAMTS8 ENSG00000134917 ADAM metallopeptidase withthrombospondin type 1 motif, 8 ADAMTS9 ENSG00000163638 ADAMmetallopeptidase with thrombospondin type 1 motif, 9 ADAMTSL1ENSG00000178031 ADAMTS-like 1 ADAMTSL2 ENSG00000197859 ADAMTS-like 2ADAMTSL3 ENSG00000156218 ADAMTS-like 3 ADAMTSL4 ENSG00000143382ADAMTS-like 4 ADAMTSL5 ENSG00000185761 ADAMTS-like 5 ADCK1ENSG00000063761 AarF domain containing kinase 1 ADCYAP1 ENSG00000141433Adenylate cyclase activating polypeptide 1 (pituitary) ADCYAP1R1ENSG00000078549 Adenylate cyclase activating polypeptide 1 (pituitary)receptor type I ADGRA3 ENSG00000152990 Adhesion G protein-coupledreceptor A3 ADGRB2 ENSG00000121753 Adhesion G protein-coupled receptorB2 ADGRD1 ENSG00000111452 Adhesion G protein-coupled receptor D1 ADGRE3ENSG00000131355 Adhesion G protein-coupled receptor E3 ADGRE5ENSG00000123146 Adhesion G protein-coupled receptor E5 ADGRF1ENSG00000153292 Adhesion G protein-coupled receptor F1 ADGRG1ENSG00000205336 Adhesion G protein-coupled receptor G1 ADGRG5ENSG00000159618 Adhesion G protein-coupled receptor G5 ADGRG6ENSG00000112414 Adhesion G protein-coupled receptor G6 ADGRV1ENSG00000164199 Adhesion G protein-coupled receptor V1 ADI1ENSG00000182551 Acireductone dioxygenase 1 ADIG ENSG00000182035Adipogenin ADIPOQ ENSG00000181092 Adiponectin, C1Q and collagen domaincontaining ADM ENSG00000148926 Adrenomedullin ADM2 ENSG00000128165Adrenomedullin 2 ADM5 ENSG00000224420 Adrenomedullin 5 (putative) ADPGKENSG00000159322 ADP-dependent glucokinase ADPRHL2 ENSG00000116863ADP-ribosylhydrolase like 2 AEBP1 ENSG00000106624 AE binding protein 1AFM ENSG00000079557 Afamin AFP ENSG00000081051 Alpha-fetoprotein AGAENSG00000038002 Aspartylglucosaminidase AGER ENSG00000204305 Advancedglycosylation end product-specific receptor AGK ENSG00000006530Acylglycerol kinase AGPS ENSG00000018510 Alkylglycerone phosphatesynthase AGR2 ENSG00000106541 Anterior gradient 2, protein disulphideisomerase family member AGR3 ENSG00000173467 Anterior gradient 3,protein disulphide isomerase family member AGRN ENSG00000188157 AgrinAGRP ENSG00000159723 Agouti related neuropeptide AGT ENSG00000135744Angiotensinogen (serpin peptidase inhibitor, clade A, member 8) AGTPBP1ENSG00000135049 ATP/GTP binding protein 1 AGTRAP ENSG00000177674Angiotensin II receptor-associated protein AHCYL2 ENSG00000158467Adenosylhomocysteinase-like 2 AHSG ENSG00000145192Alpha-2-HS-glycoprotein AIG1 ENSG00000146416 Androgen-induced 1 AK4ENSG00000162433 Adenylate kinase 4 AKAP10 ENSG00000108599 A kinase(PRKA) anchor protein 10 AKR1C1 ENSG00000187134 Aldo-keto reductasefamily 1, member Cl AL356289.1 ENSG00000279096 AL589743.1ENSG00000279508 ALAS2 ENSG00000158578 5′-aminolevulinate synthase 2 ALBENSG00000163631 Albumin ALDH9A1 ENSG00000143149 Aldehyde dehydrogenase 9family, member A1 ALDOA ENSG00000149925 Aldolase A,fructose-bisphosphate ALG1 ENSG00000033011 ALG1,chitobiosyldiphosphodolichol beta-mannosyltransferase ALG5ENSG00000120697 ALG5, dolichyl-phosphate beta-glucosyltransferase ALG9ENSG00000086848 ALG9, alpha-1,2-mannosyltransferase ALKBH1ENSG00000100601 AlkB homolog 1, histone H2A dioxygenase ALKBH5ENSG00000091542 AlkB homolog 5, RNA demethylase ALPI ENSG00000163295Alkaline phosphatase, intestinal ALPL ENSG00000162551 Alkalinephosphatase, liver/bone/kidney ALPP ENSG00000163283 Alkalinephosphatase, placental ALPPL2 ENSG00000163286 Alkaline phosphatase,placental-like 2 AMBN ENSG00000178522 Ameloblastin (enamel matrixprotein) AMBP ENSG00000106927 Alpha-1-microglobulin/bikunin precursorAMELX ENSG00000125363 Amelogenin, X-linked AMELY ENSG00000099721Amelogenin, Y-linked AMH ENSG00000104899 Anti-Mullerian hormone AMICA1ENSG00000160593 Adhesion molecule, interacts with CXADR antigen 1 AMPD1ENSG00000116748 Adenosine monophosphate deaminase 1 AMTN ENSG00000187689Amelotin AMY1A ENSG00000237763 Amylase, alpha 1A (salivary) AMY1BENSG00000174876 Amylase, alpha 1B (salivary) AMY1C ENSG00000187733Amylase, alpha 1C (salivary) AMY2A ENSG00000243480 Amylase, alpha 2A(pancreatic) AMY2B ENSG00000240038 Amylase, alpha 2B (pancreatic) ANGENSG00000214274 Angiogenin, ribonuclease, RNase A family, 5 ANGEL1ENSG00000013523 Angel homolog 1 (Drosophila) ANGPT1 ENSG00000154188Angiopoietin 1 ANGPT2 ENSG00000091879 Angiopoietin 2 ANGPT4ENSG00000101280 Angiopoietin 4 ANGPTL1 ENSG00000116194 Angiopoietin-like1 ANGPTL2 ENSG00000136859 Angiopoietin-like 2 ANGPTL3 ENSG00000132855Angiopoietin-like 3 ANGPTL4 ENSG00000167772 Angiopoietin-like 4 ANGPTL5ENSG00000187151 Angiopoietin-like 5 ANGPTL6 ENSG00000130812Angiopoietin-like 6 ANGPTL7 ENSG00000171819 Angiopoietin-like 7 ANK1ENSG00000029534 Ankyrin 1, erythrocytic ANKDD1A ENSG00000166839 Ankyrinrepeat and death domain containing lA ANKRD54 ENSG00000100124 Ankyrinrepeat domain 54 ANKRD60 ENSG00000124227 Ankyrin repeat domain 60 ANO7ENSG00000146205 Anoctamin 7 ANOS1 ENSG00000011201 Anosmin 1 ANTXR1ENSG00000169604 Anthrax toxin receptor 1 AOAH ENSG00000136250Acyloxyacyl hydrolase (neutrophil) AOC1 ENSG00000002726 Amine oxidase,copper containing 1 AOC2 ENSG00000131480 Amine oxidase, coppercontaining 2 (retina-specific) AOC3 ENSG00000131471 Amine oxidase,copper containing 3 AP000721.4 ENSG00000256100 AP000866.1ENSG00000279342 APBB1 ENSG00000166313 Amyloid beta (A4) precursorprotein-binding, family B, member 1 (Fe65) APCDD1 ENSG00000154856Adenomatosis polyposis coli down-regulated 1 APCS ENSG00000132703Amyloid P component, serum APELA ENSG00000248329 Apelin receptor earlyendogenous ligand APLN ENSG00000171388 Apelin APLP2 ENSG00000084234Amyloid beta (A4) precursor-like protein 2 APOA1 ENSG00000118137Apolipoprotein A-I APOA1BP ENSG00000163382 Apolipoprotein A-I bindingprotein APOA2 ENSG00000158874 Apolipoprotein A-II APOA4 ENSG00000110244Apolipoprotein A-IV APOA5 ENSG00000110243 Apolipoprotein A-V APOBENSG00000084674 Apolipoprotein B APOC1 ENSG00000130208 ApolipoproteinC-I APOC2 ENSG00000234906 Apolipoprotein C-II APOC3 ENSG00000110245Apolipoprotein C-III APOC4 ENSG00000267467 Apolipoprotein C-IVAPOC4-APOC2 ENSG00000224916 APOC4-APOC2 readthrough (NMD candidate) APODENSG00000189058 Apolipoprotein D APOE ENSG00000130203 Apolipoprotein EAPOF ENSG00000175336 Apolipoprotein F APOH ENSG00000091583Apolipoprotein H (beta-2-glycoprotein I) APOL1 ENSG00000100342Apolipoprotein L, 1 APOL3 ENSG00000128284 Apolipoprotein L, 3 APOMENSG00000204444 Apolipoprotein M APOOL ENSG00000155008 ApolipoproteinO-like ARCN1 ENSG00000095139 Archain 1 ARFIP2 ENSG00000132254ADP-ribosylation factor interacting protein 2 ARHGAP36 ENSG00000147256Rho GTPase activating protein 36 ARHGAP6 ENSG00000047648 Rho GTPaseactivating protein 6 ARHGEF4 ENSG00000136002 Rho guanine nucleotideexchange factor (GEF) 4 ARL16 ENSG00000214087 ADP-ribosylationfactor-like 16 ARMC5 ENSG00000140691 Armadillo repeat containing 5 ARNTLENSG00000133794 Aryl hydrocarbon receptor nuclear translocator-like ARSAENSG00000100299 Arylsulfatase A ARSB ENSG00000113273 Arylsulfatase BARSE ENSG00000157399 Arylsulfatase E (chondrodysplasia punctata 1) ARSGENSG00000141337 Arylsulfatase G ARSI ENSG00000183876 Arylsulfatasefamily, member I ARSK ENSG00000164291 Arylsulfatase family, member KART3 ENSG00000156219 ADP-ribosyltransferase 3 ART4 ENSG00000111339ADP-ribosyltransferase 4 (Dombrock blood group) ART5 ENSG00000167311ADP-ribosyltransferase 5 ARTN ENSG00000117407 Artemin ASAH1ENSG00000104763 N-acylsphingosine amidohydrolase (acid ceramidase) 1ASAH2 ENSG00000188611 N-acylsphingosine amidohydrolase (non-lysosomalceramidase) 2 ASCL1 ENSG00000139352 Achaete-scute family bHLHtranscription factor 1 ASIP ENSG00000101440 Agouti signaling proteinASPN ENSG00000106819 Asporin ASTL ENSG00000188886 Astacin-likemetallo-endopeptidase (M12 family) ATAD5 ENSG00000176208 ATPase family,AAA domain containing 5 ATAT1 ENSG00000137343 Alpha tubulinacetyltransferase 1 ATG2A ENSG00000110046 Autophagy related 2A ATG5ENSG00000057663 Autophagy related 5 ATMIN ENSG00000166454 ATM interactorATP13A1 ENSG00000105726 ATPase type 13A1 ATP5F1 ENSG00000116459 ATPsynthase, H+ transporting, mitochondrial Fo complex, subunit B1 ATP6AP1ENSG00000071553 ATPase, H+ transporting, lysosomal accessory protein 1ATP6AP2 ENSG00000182220 ATPase, H+ transporting, lysosomal accessoryprotein 2 ATPAF1 ENSG00000123472 ATP synthase mitochondrial F1 complexassembly factor 1 AUH ENSG00000148090 AU RNA binding protein/enoyl-CoAhydratase AVP ENSG00000101200 Arginine vasopressin AXIN2 ENSG00000168646Axin 2 AZGP1 ENSG00000160862 Alpha-2-glycoprotein 1, zinc-binding AZU1ENSG00000172232 Azurocidin 1 B2M ENSG00000166710 Beta-2-microglobulinB3GALNT1 ENSG00000169255 Beta-1,3-N-acetylgalactosaminyltransferase 1(globoside blood group) B3GALNT2 ENSG00000162885Beta-1,3-N-acetylgalactosaminyltransferase 2 B3GALT1 ENSG00000172318UDP-Gal:betaGlcNAc beta 1,3-galactosyltransferase, polypeptide 1 B3GALT4ENSG00000235863 UDP-Gal:betaGlcNAc beta 1,3-galactosyltransferase,polypeptide 4 B3GALT5 ENSG00000183778 UDP-Gal:betaGlcNAc beta1,3-galactosyltransferase, polypeptide 5 B3GALT6 ENSG00000176022UDP-Gal:betaGal beta 1,3-galactosyltransferase polypeptide 6 B3GAT3ENSG00000149541 Beta-1,3-glucuronyltransferase 3 B3GLCT ENSG00000187676Beta 3-glucosyltransferase B3GNT3 ENSG00000179913 UDP-GlcNAc:betaGalbeta-1,3-N-acetylglucosaminyltransferase 3 B3GNT4 ENSG00000176383UDP-GlcNAc:betaGal beta-1,3-N-acetylglucosaminyltransferase 4 B3GNT6ENSG00000198488 UDP-GlcNAc:betaGalbeta-1,3-N-acetylglucosaminyltransferase 6 B3GNT7 ENSG00000156966UDP-GlcNAc:betaGal beta-1,3-N-acetylglucosaminyltransferase 7 B3GNT8ENSG00000177191 UDP-GlcNAc:betaGalbeta-1,3-N-acetylglucosaminyltransferase 8 B3GNT9 ENSG00000237172UDP-GlcNAc:betaGal beta-1,3-N-acetylglucosaminyltransferase 9 B4GALNT1ENSG00000135454 Beta-1,4-N-acetyl-galactosaminyl transferase 1 B4GALNT3ENSG00000139044 Beta-1,4-N-acetyl-galactosaminyl transferase 3 B4GALNT4ENSG00000182272 Beta-1,4-N-acetyl-galactosaminyl transferase 4 B4GALT4ENSG00000121578 UDP-Gal:betaGlcNAc beta 1,4- galactosyltransferase,polypeptide 4 B4GALT5 ENSG00000158470 UDP-Gal:betaGlcNAc beta 1,4-galactosyltransferase, polypeptide 5 B4GALT6 ENSG00000118276UDP-Gal:betaGlcNAc beta 1,4- galactosyltransferase, polypeptide 6 B4GAT1ENSG00000174684 Beta-1,4-glucuronyltransferase 1 B9D1 ENSG00000108641 B9protein domain 1 BACE2 ENSG00000182240 Beta-site APP-cleaving enzyme 2BAGE5 ENSG00000279973 B melanoma antigen family, member 5 BCAMENSG00000187244 Basal cell adhesion molecule (Lutheran blood group) BCANENSG00000132692 Brevican BCAP29 ENSG00000075790 B-cellreceptor-associated protein 29 BCAR1 ENSG00000050820 Breast canceranti-estrogen resistance 1 BCHE ENSG00000114200 ButyrylcholinesteraseBCKDHB ENSG00000083123 Branched chain keto acid dehydrogenase E1, betapolypeptide BDNF ENSG00000176697 Brain-derived neurotrophic factor BGLAPENSG00000242252 Bone gamma-carboxyglutamate (gla) protein BGNENSG00000182492 Biglycan BLVRB ENSG00000090013 Biliverdin reductase BBMP1 ENSG00000168487 Bone morphogenetic protein 1 BMP10 ENSG00000163217Bone morphogenetic protein 10 BMP15 ENSG00000130385 Bone morphogeneticprotein 15 BMP2 ENSG00000125845 Bone morphogenetic protein 2 BMP3ENSG00000152785 Bone morphogenetic protein 3 BMP4 ENSG00000125378 Bonemorphogenetic protein 4 BMP6 ENSG00000153162 Bone morphogenetic protein6 BMP7 ENSG00000101144 Bone morphogenetic protein 7 BMP8AENSG00000183682 Bone morphogenetic protein 8a BMP8B ENSG00000116985 Bonemorphogenetic protein 8b BMPER ENSG00000164619 BMPbinding endothelialregulator BNC1 ENSG00000169594 Basonuclin 1 BOC ENSG00000144857 BOC celladhesion associated, oncogene regulated BOD1 ENSG00000145919Biorientation of chromosomes in cell division 1 BOLA1 ENSG00000178096BolA family member 1 BPI ENSG00000101425Bactericidal/permeability-increasing protein BPIFA1 ENSG00000198183 BPIfold containing family A, member 1 BPIFA2 ENSG00000131050 BPI foldcontaining family A, member 2 BPIFA3 ENSG00000131059 BPI fold containingfamily A, member 3 BPIFB1 ENSG00000125999 BPI fold containing family B,member 1 BPIFB2 ENSG00000078898 BPI fold containing family B, member 2BPIFB3 ENSG00000186190 BPI fold containing family B, member 3 BPIFB4ENSG00000186191 BPI fold containing family B, member 4 BPIFB6ENSG00000167104 BPI fold containing family B, member 6 BPIFCENSG00000184459 BPI fold containing family C BRF1 ENSG00000185024 BRF1,RNA polymerase III transcription initiation factor 90 kDa subunit BRINP1ENSG00000078725 Bone morphogenetic protein/retinoic acid inducibleneural-specific 1 BRINP2 ENSG00000198797 Bone morphogeneticprotein/retinoic acid inducible neural-specific 2 BRINP3 ENSG00000162670Bone morphogenetic protein/retinoic acid inducible neural-specific 3 BSGENSG00000172270 Basigin (Ok blood group) BSPH1 ENSG00000188334 Binder ofsperm protein homolog 1 BST1 ENSG00000109743 Bone marrow stromal cellantigen 1 BTBD17 ENSG00000204347 BTB (POZ) domain containing 17 BTDENSG00000169814 Biotinidase BTN2A2 ENSG00000124508 Butyrophilin,subfamily 2, member A2 BTN3A1 ENSG00000026950 Butyrophilin, subfamily 3,member A1 BTN3A2 ENSG00000186470 Butyrophilin, subfamily 3, member A2BTN3A3 ENSG00000111801 Butyrophilin, subfamily 3, member A3 C10orf10ENSG00000165507 Chromosome 10 open reading frame 10 C10orf99ENSG00000188373 Chromosome 10 open reading frame 99 C11orf1ENSG00000137720 Chromosome 11 open reading frame 1 C11orf24ENSG00000171067 Chromosome 11 open reading frame 24 C11orf45ENSG00000174370 Chromosome 11 open reading frame 45 C11orf94ENSG00000234776 Chromosome 11 open reading frame 94 C12orf10ENSG00000139637 Chromosome 12 open reading frame 10 C12orf49ENSG00000111412 Chromosome 12 open reading frame 49 C12orf73ENSG00000204954 Chromosome 12 open reading frame 73 C12orf76ENSG00000174456 Chromosome 12 open reading frame 76 C14orf80ENSG00000185347 Chromosome 14 open reading frame 80 C14orf93ENSG00000100802 Chromosome 14 open reading frame 93 C16orf89ENSG00000153446 Chromosome 16 open reading frame 89 C16orf90ENSG00000215131 Chromosome 16 open reading frame 90 C17orf67ENSG00000214226 Chromosome 17 open reading frame 67 C17orf75ENSG00000108666 Chromosome 17 open reading frame 75 C17orf99ENSG00000187997 Chromosome 17 open reading frame 99 C18orf54ENSG00000166845 Chromosome 18 open reading frame 54 C19orf47ENSG00000160392 Chromosome 19 open reading frame 47 C19orf70ENSG00000174917 Chromosome 19 open reading frame 70 C19orf80ENSG00000130173 Chromosome 19 open reading frame 80 C1GALT1ENSG00000106392 Core 1 synthase, glycoprotein-N-acetylgalactosamine3-beta-galactosyltransferase 1 C1orf127 ENSG00000175262 Chromosome 1open reading frame 127 C1orf159 ENSG00000131591 Chromosome 1 openreading frame 159 C1orf198 ENSG00000119280 Chromosome 1 open readingframe 198 C1orf234 ENSG00000227868 Chromosome 1 open reading frame 234C1orf54 ENSG00000118292 Chromosome 1 open reading frame 54 C1orf56ENSG00000143443 Chromosome 1 open reading frame 56 C1QA ENSG00000173372Complement component 1, q subcomponent, A chain C1QB ENSG00000173369Complement component 1, q subcomponent, B chain C1QC ENSG00000159189Complement component 1, q subcomponent, C chain C1QL1 ENSG00000131094Complement component 1, q subcomponent-like 1 C1QL2 ENSG00000144119Complement component 1, q subcomponent-like 2 C1QL3 ENSG00000165985Complement component 1, q subcomponent-like 3 C1QL4 ENSG00000186897Complement component 1, q subcomponent-like 4 C1QTNF1 ENSG00000173918C1q and tumor necrosis factor related protein 1 C1QTNF2 ENSG00000145861C1q and tumor necrosis factor related protein 2 C1QTNF3 ENSG00000082196C1q and tumor necrosis factor related protein 3 C1QTNF4 ENSG00000172247C1q and tumor necrosis factor related protein 4 C1QTNF5 ENSG00000223953C1q and tumor necrosis factor related protein 5 C1QTNF7 ENSG00000163145C1q and tumor necrosis factor related protein 7 C1QTNF8 ENSG00000184471C1q and tumor necrosis factor related protein 8 C1QTNF9 ENSG00000240654C1q and tumor necrosis factor related protein 9 C1QTNF9B ENSG00000205863C1q and tumor necrosis factor related protein 9B C1R ENSG00000159403Complement component 1, r subcomponent C1RL ENSG00000139178 Complementcomponent 1, r subcomponent-like C1S ENSG00000182326 Complementcomponent 1, s subcomponent C2 ENSG00000166278 Complement component 2C21orf33 ENSG00000160221 Chromosome 21 open reading frame 33 C21orf62ENSG00000205929 Chromosome 21 open reading frame 62 C22orf15ENSG00000169314 Chromosome 22 open reading frame 15 C22orf46ENSG00000184208 Chromosome 22 open reading frame 46 C2CD2ENSG00000157617 C2 calcium-dependent domain containing 2 C2orf40ENSG00000119147 Chromosome 2 open reading frame 40 C2orf66ENSG00000187944 Chromosome 2 open reading frame 66 C2orf69ENSG00000178074 Chromosome 2 open reading frame 69 C2orf78ENSG00000187833 Chromosome 2 open reading frame 78 C3 ENSG00000125730Complement component 3 C3orf33 ENSG00000174928 Chromosome 3 open readingframe 33 C3orf58 ENSG00000181744 Chromosome 3 open reading frame 58 C4AENSG00000244731 Complement component 4A (Rodgers blood group) C4BENSG00000224389 Complement component 4B (Chido blood group) C4BPAENSG00000123838 Complement component 4 binding protein, alpha C4BPBENSG00000123843 Complement component 4 binding protein, beta C4orf26ENSG00000174792 Chromosome 4 open reading frame 26 C4orf48ENSG00000243449 Chromosome 4 open reading frame 48 C5 ENSG00000106804Complement component 5 C5orf46 ENSG00000178776 Chromosome 5 open readingframe 46 C6 ENSG00000039537 Complement component 6 C6orf120ENSG00000185127 Chromosome 6 open reading frame 120 C6orf15ENSG00000204542 Chromosome 6 open reading frame 15 C6orf25ENSG00000204420 Chromosome 6 open reading frame 25 C6orf58ENSG00000184530 Chromosome 6 open reading frame 58 C7 ENSG00000112936Complement component 7 C7orf57 ENSG00000164746 Chromosome 7 open readingframe 57 C7orf73 ENSG00000243317 Chromosome 7 open reading frame 73 C8AENSG00000157131 Complement component 8, alpha polypeptide C8BENSG00000021852 Complement component 8, beta polypeptide C8GENSG00000176919 Complement component 8, gamma polypeptide C9ENSG00000113600 Complement component 9 C9orf47 ENSG00000186354Chromosome 9 open reading frame 47 CA10 ENSG00000154975 Carbonicanhydrase X CA11 ENSG00000063180 Carbonic anhydrase XI CA6ENSG00000131686 Carbonic anhydrase VI CA9 ENSG00000107159 Carbonicanhydrase IX CABLES1 ENSG00000134508 Cdk5 and Abl enzyme substrate 1CABP1 ENSG00000157782 Calcium binding protein 1 CACNA2D1 ENSG00000153956Calcium channel, voltage-dependent, alpha 2/delta subunit 1 CACNA2D4ENSG00000151062 Calcium channel, voltage-dependent, alpha 2/deltasubunit 4 CADM3 ENSG00000162706 Cell adhesion molecule 3 CALCAENSG00000110680 Calcitonin-related polypeptide alpha CALCBENSG00000175868 Calcitonin-related polypeptide beta CALCRENSG00000004948 Calcitonin receptor CALCRL ENSG00000064989 Calcitoninreceptor-like CALR ENSG00000179218 Calreticulin CALR3 ENSG00000269058Calreticulin 3 CALU ENSG00000128595 Calumenin CAMK2D ENSG00000145349Calcium/calmodulin-dependent protein kinase II delta CAMPENSG00000164047 Cathelicidin antimicrobial peptide CANX ENSG00000127022Calnexin CARKD ENSG00000213995 Carbohydrate kinase domain containingCARM1 ENSG00000142453 Coactivator-associated arginine methyltransferase1 CARNS1 ENSG00000172508 Carnosine synthase 1 CARTPT ENSG00000164326CART prepropeptide CASQ1 ENSG00000143318 Calsequestrin 1 (fast-twitch,skeletal muscle) CASQ2 ENSG00000118729 Calsequestrin 2 (cardiac muscle)CATSPERG ENSG00000099338 Catsper channel auxiliary subunit gamma CBLN1ENSG00000102924 Cerebellin 1 precursor CBLN2 ENSG00000141668 Cerebellin2 precursor CBLN3 ENSG00000139899 Cerebellin 3 precursor CBLN4ENSG00000054803 Cerebellin 4 precursor CCBE1 ENSG00000183287 Collagenand calcium binding EGF domains 1 CCDC108 ENSG00000181378 Coiled-coildomain containing 108 CCDC112 ENSG00000164221 Coiled-coil domaincontaining 112 CCDC129 ENSG00000180347 Coiled-coil domain containing 129CCDC134 ENSG00000100147 Coiled-coil domain containing 134 CCDC149ENSG00000181982 Coiled-coil domain containing 149 CCDC3 ENSG00000151468Coiled-coil domain containing 3 CCDC80 ENSG00000091986 Coiled-coildomain containing 80 CCDC85A ENSG00000055813 Coiled-coil domaincontaining 85A CCDC88B ENSG00000168071 Coiled-coil domain containing 88BCCER2 ENSG00000262484 Coiled-coil glutamate-rich protein 2 CCKENSG00000187094 Cholecystokinin CCL1 ENSG00000108702 Chemokine (C-Cmotif) ligand 1 CCL11 ENSG00000172156 Chemokine (C-C motif) ligand 11CCL13 ENSG00000181374 Chemokine (C-C motif) ligand 13 CCL14ENSG00000276409 Chemokine (C-C motif) ligand 14 CCL15 ENSG00000275718Chemokine (C-C motif) ligand 15 CCL16 ENSG00000275152 Chemokine (C-Cmotif) ligand 16 CCL17 ENSG00000102970 Chemokine (C-C motif) ligand 17CCL18 ENSG00000275385 Chemokine (C-C motif) ligand 18 (pulmonary andactivation-regulated) CCL19 ENSG00000172724 Chemokine (C-C motif) ligand19 CCL2 ENSG00000108691 Chemokine (C-C motif) ligand 2 CCL20ENSG00000115009 Chemokine (C-C motif) ligand 20 CCL21 ENSG00000137077Chemokine (C-C motif) ligand 21 CCL22 ENSG00000102962 Chemokine (C-Cmotif) ligand 22 CCL23 ENSG00000274736 Chemokine (C-C motif) ligand 23CCL24 ENSG00000106178 Chemokine (C-C motif) ligand 24 CCL25ENSG00000131142 Chemokine (C-C motif) ligand 25 CCL26 ENSG00000006606Chemokine (C-C motif) ligand 26 CCL27 ENSG00000213927 Chemokine (C-Cmotif) ligand 27 CCL28 ENSG00000151882 Chemokine (C-C motif) ligand 28CCL3 ENSG00000277632 Chemokine (C-C motif) ligand 3 CCL3L3ENSG00000276085 Chemokine (C-C motif) ligand 3-like 3 CCL4ENSG00000275302 Chemokine (C-C motif) ligand 4 CCL4L2 ENSG00000276070Chemokine (C-C motif) ligand 4-like 2 CCL5 ENSG00000271503 Chemokine(C-C motif) ligand 5 CCL7 ENSG00000108688 Chemokine (C-C motif) ligand 7CCL8 ENSG00000108700 Chemokine (C-C motif) ligand 8 CCNB11P1ENSG00000100814 Cyclin B1 interacting protein 1, E3 ubiquitin proteinligase CCNL1 ENSG00000163660 Cyclin L1 CCNL2 ENSG00000221978 Cyclin L2CD14 ENSG00000170458 CD14 molecule CD160 ENSG00000117281 CD160 moleculeCD164 ENSG00000135535 CD164 molecule, sialomucin CD177 ENSG00000204936CD177 molecule CD1E ENSG00000158488 CD1e molecule CD2 ENSG00000116824CD2 molecule CD200 ENSG00000091972 CD200 molecule CD200R1ENSG00000163606 CD200 receptor 1 CD22 ENSG00000012124 CD22 moleculeCD226 ENSG00000150637 CD226 molecule CD24 ENSG00000272398 CD24 moleculeCD276 ENSG00000103855 CD276 molecule CD300A ENSG00000167851 CD300amolecule CD300LB ENSG00000178789 CD300 molecule-like family member bCD300LF ENSG00000186074 CD300 molecule-like family member f CD300LGENSG00000161649 CD300 molecule-like family member g CD3D ENSG00000167286CD3d molecule, delta (CD3-TCR complex) CD4 ENSG00000010610 CD4 moleculeCD40 ENSG00000101017 CD40 molecule, TNF receptor superfamily member 5CD44 ENSG00000026508 CD44 molecule (Indian blood group) CD48ENSG00000117091 CD48 molecule CD5 ENSG00000110448 CD5 molecule CD55ENSG00000196352 CD55 molecule, decay accelerating factor for complement(Cromer blood group) CD59 ENSG00000085063 CD59 molecule, complementregulatory protein CD5L ENSG00000073754 CD5 molecule-like CD6ENSG00000013725 CD6 molecule CD68 ENSG00000129226 CD68 molecule CD7ENSG00000173762 CD7 molecule CD79A ENSG00000105369 CD79a molecule,immunoglobulin-associated alpha CD80 ENSG00000121594 CD80 molecule CD86ENSG00000114013 CD86 molecule CD8A ENSG00000153563 CD8a molecule CD8BENSG00000172116 CD8b molecule CD99 ENSG00000002586 CD99 molecule CDC23ENSG00000094880 Cell division cycle 23 CDC40 ENSG00000168438 Celldivision cycle 40 CDC45 ENSG00000093009 Cell division cycle 45 CDCP1ENSG00000163814 CUB domain containing protein 1 CDCP2 ENSG00000157211CUB domain containing protein 2 CDH1 ENSG00000039068 Cadherin 1, type 1CDH11 ENSG00000140937 Cadherin 11, type 2, OB-cadherin (osteoblast)CDH13 ENSG00000140945 Cadherin 13 CDH17 ENSG00000079112 Cadherin 17, LIcadherin (liver-intestine) CDH18 ENSG00000145526 Cadherin 18, type 2CDH19 ENSG00000071991 Cadherin 19, type 2 CDH23 ENSG00000107736Cadherin-related 23 CDH5 ENSG00000179776 Cadherin 5, type 2 (vascularendothelium) CDHR1 ENSG00000148600 Cadherin-related family member 1CDHR4 ENSG00000187492 Cadherin-related family member 4 CDHR5ENSG00000099834 Cadherin-related family member 5 CDKN2A ENSG00000147889Cyclin-dependent kinase inhibitor 2A CDNF ENSG00000185267 Cerebraldopamine neurotrophic factor CDON ENSG00000064309 Cell adhesionassociated, oncogene regulated CDSN ENSG00000204539 CorneodesmosinCEACAM16 ENSG00000213892 Carcinoembryonic antigen-related cell adhesionmolecule 16 CEACAM18 ENSG00000213822 Carcinoembryonic antigen-relatedcell adhesion molecule 18 CEACAM19 ENSG00000186567 Carcinoembryonicantigen-related cell adhesion molecule 19 CEACAM5 ENSG00000105388Carcinoembryonic antigen-related cell adhesion molecule 5 CEACAM7ENSG00000007306 Carcinoembryonic antigen-related cell adhesion molecule7 CEACAM8 ENSG00000124469 Carcinoembryonic antigen-related cell adhesionmolecule 8 CECR1 ENSG00000093072 Cat eye syndrome chromosome region,candidate 1 CECR5 ENSG00000069998 Cat eye syndrome chromosome region,candidate 5 CEL ENSG00000170835 Carboxyl ester lipase CELA2AENSG00000142615 Chymotrypsin-like elastase family, member 2A CELA2BENSG00000215704 Chymotrypsin-like elastase family, member 2B CELA3AENSG00000142789 Chymotrypsin-like elastase family, member 3A CELA3BENSG00000219073 Chymotrypsin-like elastase family, member 3B CEMIPENSG00000103888 Cell migration inducing protein, hyaluronan bindingCEP89 ENSG00000121289 Centrosomal protein 89 kDa CER1 ENSG00000147869Cerberus 1, DAN family BMP antagonist CERCAM ENSG00000167123 Cerebralendothelial cell adhesion molecule CERS1 ENSG00000223802 Ceramidesynthase 1 CES1 ENSG00000198848 Carboxylesterase 1 CES3 ENSG00000172828Carboxylesterase 3 CES4A ENSG00000172824 Carboxylesterase 4A CES5AENSG00000159398 Carboxylesterase 5A CETP ENSG00000087237 Cholesterylester transfer protein, plasma CFB ENSG00000243649 Complement factor BCFC1 ENSG00000136698 Cripto, FRL-1, cryptic family 1 CFC1BENSG00000152093 Cripto, FRL-1, cryptic family 1B CFD ENSG00000197766Complement factor D (adipsin) CFDP1 ENSG00000153774 Craniofacialdevelopment protein 1 CFH ENSG00000000971 Complement factor H CFHR1ENSG00000244414 Complement factor H-related 1 CFHR2 ENSG00000080910Complement factor H-related 2 CFHR3 ENSG00000116785 Complement factorH-related 3 CFHR4 ENSG00000134365 Complement factor H-related 4 CFHR5ENSG00000134389 Complement factor H-related 5 CFI ENSG00000205403Complement factor I CFP ENSG00000126759 Complement factor properdin CGAENSG00000135346 Glycoprotein hormones, alpha polypeptide CGBENSG00000104827 Chorionic gonadotropin, beta polypeptide CGB1ENSG00000267631 Chorionic gonadotropin, beta polypeptide 1 CGB2ENSG00000104818 Chorionic gonadotropin, beta polypeptide 2 CGB5ENSG00000189052 Chorionic gonadotropin, beta polypeptide 5 CGB7ENSG00000196337 Chorionic gonadotropin, beta polypeptide 7 CGB8ENSG00000213030 Chorionic gonadotropin, beta polypeptide 8 CGREF1ENSG00000138028 Cell growth regulator with EF-hand domain 1 CH507-9B2.3ENSG00000280071 CHAD ENSG00000136457 Chondroadherin CHADLENSG00000100399 Chondroadherin-like CHEK2 ENSG00000183765 Checkpointkinase 2 CHGA ENSG00000100604 Chromogranin A CHGB ENSG00000089199Chromogranin B CHI3L1 ENSG00000133048 Chitinase 3-like 1 (cartilageglycoprotein-39) CHI3L2 ENSG00000064886 Chitinase 3-like 2 CHIAENSG00000134216 Chitinase, acidic CHID1 ENSG00000177830 Chitinase domaincontaining 1 CHIT1 ENSG00000133063 Chitinase 1 (chitotriosidase) CHL1ENSG00000134121 Cell adhesion molecule L1-like CHN1 ENSG00000128656Chimerin 1 CHPF ENSG00000123989 Chondroitin polymerizing factor CHPF2ENSG00000033100 Chondroitin polymerizing factor 2 CHRD ENSG00000090539Chordin CHRDL1 ENSG00000101938 Chordin-like 1 CHRDL2 ENSG00000054938Chordin-like 2 CHRNA2 ENSG00000120903 Cholinergic receptor, nicotinic,alpha 2 (neuronal) CHRNA5 ENSG00000169684 Cholinergic receptor,nicotinic, alpha 5 (neuronal) CHRNB1 ENSG00000170175 Cholinergicreceptor, nicotinic, beta 1 (muscle) CHRND ENSG00000135902 Cholinergicreceptor, nicotinic, delta (muscle) CHST1 ENSG00000175264 Carbohydrate(keratan sulfate Gal-6) sulfotransferase 1 CHST10 ENSG00000115526Carbohydrate sulfotransferase 10 CHST11 ENSG00000171310 Carbohydrate(chondroitin 4) sulfotransferase 11 CHST13 ENSG00000180767 Carbohydrate(chondroitin 4) sulfotransferase 13 CHST4 ENSG00000140835 Carbohydrate(N-acetylglucosamine 6-0) sulfotransferase 4 CHST5 ENSG00000135702Carbohydrate (N-acetylglucosamine 6-0) sulfotransferase 5 CHST6ENSG00000183196 Carbohydrate (N-acetylglucosamine 6-0) sulfotransferase6 CHST7 ENSG00000147119 Carbohydrate (N-acetylglucosamine 6-0)sulfotransferase 7 CHST8 ENSG00000124302 Carbohydrate(N-acetylgalactosamine 4-0) sulfotransferase 8 CHSY1 ENSG00000131873Chondroitin sulfate synthase 1 CHSY3 ENSG00000198108 Chondroitin sulfatesynthase 3 CHTF8 ENSG00000168802 Chromosome transmission fidelity factor8 CILP ENSG00000138615 Cartilage intermediate layer protein, nucleotidepyrophosphohydrolase CILP2 ENSG00000160161 Cartilage intermediate layerprotein 2 CIRH1A ENSG00000141076 Cirrhosis, autosomal recessive lA(cirhin) CKLF ENSG00000217555 Chemokine-like factor CKMT1AENSG00000223572 Creatine kinase, mitochondrial lA CKMT1B ENSG00000237289Creatine kinase, mitochondrial 1B CLCA1 ENSG00000016490 Chloride channelaccessory 1 CLCF1 ENSG00000175505 Cardiotrophin-like cytokine factor 1CLDN15 ENSG00000106404 Claudin 15 CLDN7 ENSG00000181885 Claudin 7 CLDND1ENSG00000080822 Claudin domain containing 1 CLEC11A ENSG00000105472C-type lectin domain family 11, member A CLEC16A ENSG00000038532 C-typelectin domain family 16, member A CLEC18A ENSG00000157322 C-type lectindomain family 18, member A CLEC18B ENSG00000140839 C-type lectin domainfamily 18, member B CLEC18C ENSG00000157335 C-type lectin domain family18, member C CLEC19A ENSG00000261210 C-type lectin domain family 19,member A CLEC2B ENSG00000110852 C-type lectin domain family 2, member BCLEC3A ENSG00000166509 C-type lectin domain family 3, member A CLEC3BENSG00000163815 C-type lectin domain family 3, member B CLGNENSG00000153132 Calmegin CLN5 ENSG00000102805 Ceroid-lipofuscinosis,neuronal 5 CLPS ENSG00000137392 Colipase, pancreatic CLPSL1ENSG00000204140 Colipase-like 1 CLPSL2 ENSG00000196748 Colipase-like 2CLPX ENSG00000166855 Caseinolytic mitochondrial matrix peptidasechaperone subunit CLSTN3 ENSG00000139182 Calsyntenin 3 CLUENSG00000120885 Clusterin CLUL1 ENSG00000079101 Clusterin-like 1(retinal) CMA1 ENSG00000092009 Chymase 1, mast cell CMPK1ENSG00000162368 Cytidine monophosphate (UMP-CMP) kinase 1, cytosolicCNBD1 ENSG00000176571 Cyclic nucleotide binding domain containing 1CNDP1 ENSG00000150656 Carnosine dipeptidase 1 (metallopeptidase M20family) CNPY2 ENSG00000257727 Canopy FGF signaling regulator 2 CNPY3ENSG00000137161 Canopy FGF signaling regulator 3 CNPY4 ENSG00000166997Canopy FGF signaling regulator 4 CNTFR ENSG00000122756 Ciliaryneurotrophic factor receptor CNTN1 ENSG00000018236 Contactin 1 CNTN2ENSG00000184144 Contactin 2 (axonal) CNTN3 ENSG00000113805 Contactin 3(plasmacytoma associated) CNTN4 ENSG00000144619 Contactin 4 CNTN5ENSG00000149972 Contactin 5 CNTNAP2 ENSG00000174469 Contactin associatedprotein-like 2 CNTNAP3 ENSG00000106714 Contactin associated protein-like3 CNTNAP3B ENSG00000154529 Contactin associated protein-like 3B COASYENSG00000068120 CoA synthase COCH ENSG00000100473 Cochlin COG3ENSG00000136152 Component of oligomeric golgi complex 3 COL10A1ENSG00000123500 Collagen, type X, alpha 1 COL11A1 ENSG00000060718Collagen, type XI, alpha 1 COL11A2 ENSG00000204248 Collagen, type XI,alpha 2 COL12A1 ENSG00000111799 Collagen, type XII, alpha 1 COL14A1ENSG00000187955 Collagen, type XIV, alpha 1 COL15A1 ENSG00000204291Collagen, type XV, alpha 1 COL16A1 ENSG00000084636 Collagen, type XVI,alpha 1 COL18A1 ENSG00000182871 Collagen, type XVIII, alpha 1 COL19A1ENSG00000082293 Collagen, type XIX, alpha 1 COL1A1 ENSG00000108821Collagen, type I, alpha 1 COL1A2 ENSG00000164692 Collagen, type I, alpha2 COL20A1 ENSG00000101203 Collagen, type XX, alpha 1 COL21A1ENSG00000124749 Collagen, type XXI, alpha 1 COL22A1 ENSG00000169436Collagen, type XXII, alpha 1 COL24A1 ENSG00000171502 Collagen, typeXXIV, alpha 1 COL26A1 ENSG00000160963 Collagen, type XXVI, alpha 1COL27A1 ENSG00000196739 Collagen, type XXVII, alpha 1 COL28A1ENSG00000215018 Collagen, type XXVIII, alpha 1 COL2A1 ENSG00000139219Collagen, type II, alpha 1 COL3A1 ENSG00000168542 Collagen, type III,alpha 1 COL4A1 ENSG00000187498 Collagen, type IV, alpha 1 COL4A2ENSG00000134871 Collagen, type IV, alpha 2 COL4A3 ENSG00000169031Collagen, type IV, alpha 3 (Goodpasture antigen) COL4A4 ENSG00000081052Collagen, type IV, alpha 4 COL4A5 ENSG00000188153 Collagen, type IV,alpha 5 COL4A6 ENSG00000197565 Collagen, type IV, alpha 6 COL5A1ENSG00000130635 Collagen, type V, alpha 1 COL5A2 ENSG00000204262Collagen, type V, alpha 2 COL5A3 ENSG00000080573 Collagen, type V, alpha3 COL6A1 ENSG00000142156 Collagen, type VI, alpha 1 COL6A2ENSG00000142173 Collagen, type VI, alpha 2 COL6A3 ENSG00000163359Collagen, type VI, alpha 3 COL6A5 ENSG00000172752 Collagen, type VI,alpha 5 COL6A6 ENSG00000206384 Collagen, type VI, alpha 6 COL7A1ENSG00000114270 Collagen, type VII, alpha 1 COL8A1 ENSG00000144810Collagen, type VIII, alpha 1 COL8A2 ENSG00000171812 Collagen, type VIII,alpha 2 COL9A1 ENSG00000112280 Collagen, type IX, alpha 1 COL9A2ENSG00000049089 Collagen, type IX, alpha 2 COL9A3 ENSG00000092758Collagen, type IX, alpha 3 COLEC10 ENSG00000184374 Collectin sub-familymember 10 (C-type lectin) COLEC11 ENSG00000118004 Collectin sub-familymember 11 COLGALT1 ENSG00000130309 Collagenbeta(1-0)galactosyltransferase 1 COLGALT2 ENSG00000198756 Collagenbeta(1-0)galactosyltransferase 2 COLQ ENSG00000206561 Collagen-like tailsubunit (single strand of homotrimer) of asymmetric acetylcholinesteraseCOMP ENSG00000105664 Cartilage oligomeric matrix protein COPS6ENSG00000168090 COP9 signalosome subunit 6 COQ6 ENSG00000119723 CoenzymeQ6 monooxygenase CORT ENSG00000241563 Cortistatin CP ENSG00000047457Ceruloplasmin (ferroxidase) CPA1 ENSG00000091704 Carboxypeptidase A1(pancreatic) CPA2 ENSG00000158516 Carboxypeptidase A2 (pancreatic) CPA3ENSG00000163751 Carboxypeptidase A3 (mast cell) CPA4 ENSG00000128510Carboxypeptidase A4 CPA6 ENSG00000165078 Carboxypeptidase A6 CPAMD8ENSG00000160111 C3 and PZP-like, alpha-2-macroglobulin domain containing8 CPB1 ENSG00000153002 Carboxypeptidase B1 (tissue) CPB2 ENSG00000080618Carboxypeptidase B2 (plasma) CPE ENSG00000109472 Carboxypeptidase E CPMENSG00000135678 Carboxypeptidase M CPN1 ENSG00000120054 CarboxypeptidaseN, polypeptide 1 CPN2 ENSG00000178772 Carboxypeptidase N, polypeptide 2CPO ENSG00000144410 Carboxypeptidase O CPQ ENSG00000104324Carboxypeptidase Q CPVL ENSG00000106066 Carboxypeptidase,vitellogenic-like CPXM1 ENSG00000088882 Carboxypeptidase X (M14 family),member 1 CPXM2 ENSG00000121898 Carboxypeptidase X (M14 family), member 2CPZ ENSG00000109625 Carboxypeptidase Z CR1L ENSG00000197721 Complementcomponent (3b/4b) receptor 1-like CRB2 ENSG00000148204 Crumbs familymember 2 CREG1 ENSG00000143162 Cellular repressor of E1A-stimulatedgenes 1 CREG2 ENSG00000175874 Cellular repressor of E1A-stimulated genes2 CRELD1 ENSG00000163703 Cysteine-rich with EGF-like domains 1 CRELD2ENSG00000184164 Cysteine-rich with EGF-like domains 2 CRHENSG00000147571 Corticotropin releasing hormone CRHBP ENSG00000145708Corticotropin releasing hormone binding protein CRHR1 ENSG00000120088Corticotropin releasing hormone receptor 1 CRHR2 ENSG00000106113Corticotropin releasing hormone receptor 2 CRISP1 ENSG00000124812Cysteine-rich secretory protein 1 CRISP2 ENSG00000124490 Cysteine-richsecretory protein 2 CRISP3 ENSG00000096006 Cysteine-rich secretoryprotein 3 CRISPLD2 ENSG00000103196 Cysteine-rich secretory protein LCCLdomain containing 2 CRLF1 ENSG00000006016 Cytokine receptor-like factor1 CRP ENSG00000132693 C-reactive protein, pentraxin-related CRTAC1ENSG00000095713 Cartilage acidic protein 1 CRTAP ENSG00000170275Cartilage associated protein CRY2 ENSG00000121671 Cryptochrome circadianclock 2 CSAD ENSG00000139631 Cysteine sulfinic acid decarboxylase CSF1ENSG00000184371 Colony stimulating factor 1 (macrophage) CSF1RENSG00000182578 Colony stimulating factor 1 receptor CSF2ENSG00000164400 Colony stimulating factor 2 (granulocyte-macrophage)CSF2RA ENSG00000198223 Colony stimulating factor 2 receptor, alpha,low-affinity (granulocyte-macrophage) CSF3 ENSG00000108342 Colonystimulating factor 3 (granulocyte) CSGALNACT1 ENSG00000147408Chondroitin sulfate N-acetylgalactosaminyltransferase 1 CSH1ENSG00000136488 Chorionic somatomammotropin hormone 1 (placentallactogen) CSH2 ENSG00000213218 Chorionic somatomammotropin hormone 2CSHL1 ENSG00000204414 Chorionic somatomammotropin hormone-like 1 CSN1S1ENSG00000126545 Casein alpha sl CSN2 ENSG00000135222 Casein beta CSN3ENSG00000171209 Casein kappa CST1 ENSG00000170373 Cystatin SN CST11ENSG00000125831 Cystatin 11 CST2 ENSG00000170369 Cystatin SA CST3ENSG00000101439 Cystatin C CST4 ENSG00000101441 Cystatin S CST5ENSG00000170367 Cystatin D CST6 ENSG00000175315 Cystatin E/M CST7ENSG00000077984 Cystatin F (leukocystatin) CST8 ENSG00000125815 Cystatin8 (cystatin-related epididymal specific) CST9 ENSG00000173335 Cystatin 9(testatin) CST9L ENSG00000101435 Cystatin 9-like CSTL1 ENSG00000125823Cystatin-like 1 CT55 ENSG00000169551 Cancer/testis antigen 55CTB-60B18.6 ENSG00000267335 CTBS ENSG00000117151 Chitobiase,di-N-acetyl- CTD-2313N18.7 ENSG00000225805 CTD-2370N5.3 ENSG00000265118CTGF ENSG00000118523 Connective tissue growth factor CTHRC1ENSG00000164932 Collagen triple helix repeat containing 1 CTLA4ENSG00000163599 Cytotoxic T-lymphocyte-associated protein 4 CTNSENSG00000040531 Cystinosin, lysosomal cystine transporter CTRB1ENSG00000168925 Chymotrypsinogen B1 CTRB2 ENSG00000168928Chymotrypsinogen B2 CTRC ENSG00000162438 Chymotrypsin C (caldecrin) CTRLENSG00000141086 Chymotrypsin-like CTSA ENSG00000064601 Cathepsin A CTSBENSG00000164733 Cathepsin B CTSC ENSG00000109861 Cathepsin C CTSDENSG00000117984 Cathepsin D CTSE ENSG00000196188 Cathepsin E CTSFENSG00000174080 Cathepsin F CTSG ENSG00000100448 Cathepsin G CTSHENSG00000103811 Cathepsin H CTSK ENSG00000143387 Cathepsin K CTSLENSG00000135047 Cathepsin L CTSO ENSG00000256043 Cathepsin O CTSSENSG00000163131 Cathepsin S CTSV ENSG00000136943 Cathepsin V CTSWENSG00000172543 Cathepsin W CTSZ ENSG00000101160 Cathepsin Z CUBNENSG00000107611 Cubilin (intrinsic factor-cobalamin receptor) CUTAENSG00000112514 CutA divalent cation tolerance homolog (E. coli) CX3CL1ENSG00000006210 Chemokine (C-X3-C motif) ligand 1 CXADR ENSG00000154639Coxsackie virus and adenovirus receptor CXCL1 ENSG00000163739 Chemokine(C-X-C motif) ligand 1 (melanoma growth stimulating activity, alpha)CXCL10 ENSG00000169245 Chemokine (C-X-C motif) ligand 10 CXCL11ENSG00000169248 Chemokine (C-X-C motif) ligand 11 CXCL12 ENSG00000107562Chemokine (C-X-C motif) ligand 12 CXCL13 ENSG00000156234 Chemokine(C-X-C motif) ligand 13 CXCL14 ENSG00000145824 Chemokine (C-X-C motif)ligand 14 CXCL17 ENSG00000189377 Chemokine (C-X-C motif) ligand 17 CXCL2ENSG00000081041 Chemokine (C-X-C motif) ligand 2 CXCL3 ENSG00000163734Chemokine (C-X-C motif) ligand 3 CXCL5 ENSG00000163735 Chemokine (C-X-Cmotif) ligand 5 CXCL6 ENSG00000124875 Chemokine (C-X-C motif) ligand 6CXCL8 ENSG00000169429 Chemokine (C-X-C motif) ligand 8 CXCL9ENSG00000138755 Chemokine (C-X-C motif) ligand 9 CXorf36 ENSG00000147113Chromosome X open reading frame 36 CYB5D2 ENSG00000167740 Cytochrome b5domain containing 2 CYHR1 ENSG00000187954 Cysteine/histidine-rich 1CYP17A1 ENSG00000148795 Cytochrome P450, family 17, subfamily A,polypeptide 1 CYP20A1 ENSG00000119004 Cytochrome P450, family 20,subfamily A, polypeptide 1 CYP21A2 ENSG00000231852 Cytochrome P450,family 21, subfamily A, polypeptide 2 CYP26B1 ENSG00000003137 CytochromeP450, family 26, subfamily B, polypeptide 1 CYP2A6 ENSG00000255974Cytochrome P450, family 2, subfamily A, polypeptide 6 CYP2A7ENSG00000198077 Cytochrome P450, family 2, subfamily A, polypeptide 7CYP2B6 ENSG00000197408 Cytochrome P450, family 2, subfamily B,polypeptide 6 CYP2C18 ENSG00000108242 Cytochrome P450, family 2,subfamily C, polypeptide 18 CYP2C19 ENSG00000165841 Cytochrome P450,family 2, subfamily C, polypeptide 19 CYP2C8 ENSG00000138115 CytochromeP450, family 2, subfamily C, polypeptide 8 CYP2C9 ENSG00000138109Cytochrome P450, family 2, subfamily C, polypeptide 9 CYP2E1ENSG00000130649 Cytochrome P450, family 2, subfamily E, polypeptide 1CYP2F1 ENSG00000197446 Cytochrome P450, family 2, subfamily F,polypeptide 1 CYP2J2 ENSG00000134716 Cytochrome P450, family 2,subfamily J, polypeptide 2 CYP2R1 ENSG00000186104 Cytochrome P450,family 2, subfamily R, polypeptide 1 CYP2S1 ENSG00000167600 CytochromeP450, family 2, subfamily S, polypeptide 1 CYP2W1 ENSG00000073067Cytochrome P450, family 2, subfamily W, polypeptide 1 CYP46A1ENSG00000036530 Cytochrome P450, family 46, subfamily A, polypeptide 1CYP4F11 ENSG00000171903 Cytochrome P450, family 4, subfamily F,polypeptide 11 CYP4F2 ENSG00000186115 Cytochrome P450, family 4,subfamily F, polypeptide 2 CYR61 ENSG00000142871 Cysteine-rich,angiogenic inducer, 61 CYTL1 ENSG00000170891 Cytokine-like 1 D2HGDHENSG00000180902 D-2-hydroxyglutarate dehydrogenase DAG1 ENSG00000173402Dystroglycan 1 (dystrophin-associated glycoprotein 1) DAND5ENSG00000179284 DAN domain family member 5, BMP antagonist DAGENSG00000110887 D-amino-acid oxidase DAZAP2 ENSG00000183283 DAZassociated protein 2 DBH ENSG00000123454 Dopamine beta-hydroxylase(dopamine beta-monooxygenase) DBNL ENSG00000136279 Drebrin-like DCDENSG00000161634 Dermcidin DCN ENSG00000011465 Decorin DDIASENSG00000165490 DNA damage-induced apoptosis suppressor DDOSTENSG00000244038 Dolichyl-diphosphooligosaccharide--proteinglycosyltransferase subunit (non-catalytic) DDR1 ENSG00000204580Discoidin domain receptor tyrosine kinase 1 DDR2 ENSG00000162733Discoidin domain receptor tyrosine kinase 2 DDT ENSG00000099977D-dopachrome tautomerase DDX17 ENSG00000100201 DEAD (Asp-Glu-Ala-Asp)box helicase 17 DDX20 ENSG00000064703 DEAD (Asp-Glu-Ala-Asp) boxpolypeptide 20 DDX25 ENSG00000109832 DEAD (Asp-Glu-Ala-Asp) box helicase25 DDX28 ENSG00000182810 DEAD (Asp-Glu-Ala-Asp) box polypeptide 28 DEAF1ENSG00000177030 DEAF1 transcription factor DEF8 ENSG00000140995Differentially expressed in FDCP 8 homolog (mouse) DEFA1 ENSG00000206047Defensin, alpha 1 DEFA1B ENSG00000240247 Defensin, alpha 1B DEFA3ENSG00000239839 Defensin, alpha 3, neutrophil-specific DEFA4ENSG00000164821 Defensin, alpha 4, corticostatin DEFA5 ENSG00000164816Defensin, alpha 5, Paneth cell-specific DEFA6 ENSG00000164822 Defensin,alpha 6, Paneth cell-specific DEFB1 ENSG00000164825 Defensin, beta 1DEFB103A ENSG00000176797 Defensin, beta 103A DEFB103B ENSG00000177243Defensin, beta 103B DEFB104A ENSG00000176782 Defensin, beta 104ADEFB104B ENSG00000177023 Defensin, beta 104B DEFB105A ENSG00000186562Defensin, beta 105A DEFB105B ENSG00000186599 Defensin, beta 105BDEFB106A ENSG00000186579 Defensin, beta 106A DEFB106B ENSG00000187082Defensin, beta 106B DEFB107A ENSG00000186572 Defensin, beta 107ADEFB107B ENSG00000198129 Defensin, beta 107B DEFB108B ENSG00000184276Defensin, beta 108B DEFB110 ENSG00000203970 Defensin, beta 110 DEFB113ENSG00000214642 Defensin, beta 113 DEFB114 ENSG00000177684 Defensin,beta 114 DEFB115 ENSG00000215547 Defensin, beta 115 DEFB116ENSG00000215545 Defensin, beta 116 DEFB118 ENSG00000131068 Defensin,beta 118 DEFB119 ENSG00000180483 Defensin, beta 119 DEFB121ENSG00000204548 Defensin, beta 121 DEFB123 ENSG00000180424 Defensin,beta 123 DEFB124 ENSG00000180383 Defensin, beta 124 DEFB125ENSG00000178591 Defensin, beta 125 DEFB126 ENSG00000125788 Defensin,beta 126 DEFB127 ENSG00000088782 Defensin, beta 127 DEFB128ENSG00000185982 Defensin, beta 128 DEFB129 ENSG00000125903 Defensin,beta 129 DEFB130 ENSG00000232948 Defensin, beta 130 DEFB131ENSG00000186146 Defensin, beta 131 DEFB132 ENSG00000186458 Defensin,beta 132 DEFB133 ENSG00000214643 Defensin, beta 133 DEFB134ENSG00000205882 Defensin, beta 134 DEFB135 ENSG00000205883 Defensin,beta 135 DEFB136 ENSG00000205884 Defensin, beta 136 DEFB4AENSG00000171711 Defensin, beta 4A DEFB4B ENSG00000177257 Defensin, beta4B DFNA5 ENSG00000105928 Deafness, autosomal dominant 5 DFNB31ENSG00000095397 Deafness, autosomal recessive 31 DGCR2 ENSG00000070413DiGeorge syndrome critical region gene 2 DHH ENSG00000139549 Deserthedgehog DHRS4 ENSG00000157326 Dehydrogenase/reductase (SDR family)member 4 DHRS4L2 ENSG00000187630 Dehydrogenase/reductase (SDR family)member 4 like 2 DHRS7 ENSG00000100612 Dehydrogenase/reductase (SDRfamily) member 7 DHRS7C ENSG00000184544 Dehydrogenase/reductase (SDRfamily) member 7C DHRS9 ENSG00000073737 Dehydrogenase/reductase (SDRfamily) member 9 DHRSX ENSG00000169084 Dehydrogenase/reductase (SDRfamily) X-linked DHX29 ENSG00000067248 DEAH (Asp-Glu-Ala-His) boxpolypeptide 29 DHX30 ENSG00000132153 DEAH (Asp-Glu-Ala-His) box helicase30 DHX8 ENSG00000067596 DEAH (Asp-Glu-Ala-His) box polypeptide 8 DIO2ENSG00000211448 Deiodinase, iodothyronine, type II DIXDC1ENSG00000150764 DIX domain containing 1 DKK1 ENSG00000107984 DickkopfWNT signaling pathway inhibitor 1 DKK2 ENSG00000155011 Dickkopf WNTsignaling pathway inhibitor 2 DKK3 ENSG00000050165 Dickkopf WNTsignaling pathway inhibitor 3 DKK4 ENSG00000104371 Dickkopf WNTsignaling pathway inhibitor 4 DKKL1 ENSG00000104901 Dickkopf-like 1 DLG4ENSG00000132535 Discs, large homolog 4 (Drosophila) DLK1 ENSG00000185559Delta-like 1 homolog (Drosophila) DLL1 ENSG00000198719 Delta-like 1(Drosophila) DLL3 ENSG00000090932 Delta-like 3 (Drosophila) DMBT1ENSG00000187908 Deleted in malignant brain tumors 1 DMKN ENSG00000161249Dermokine DMP1 ENSG00000152592 Dentin matrix acidic phosphoprotein 1DMRTA2 ENSG00000142700 DMRT-like family A2 DNAAF5 ENSG00000164818Dynein, axonemal, assembly factor 5 DNAH14 ENSG00000185842 Dynein,axonemal, heavy chain 14 DNAJB11 ENSG00000090520 DnaJ (Hsp40) homolog,subfamily B, member 11 DNAJB9 ENSG00000128590 DnaJ (Hsp40) homolog,subfamily B, member 9 DNAJC25-GNG10 ENSG00000244115 DNAJC25-GNG10readthrough DNAJC3 ENSG00000102580 DnaJ (Hsp40) homolog, subfamily C,member 3 DNASE1 ENSG00000213918 Deoxyribonuclease I DNASE1L1ENSG00000013563 Deoxyribonuclease I-like 1 DNASE1L2 ENSG00000167968Deoxyribonuclease I-like 2 DNASE1L3 ENSG00000163687 DeoxyribonucleaseI-like 3 DNASE2 ENSG00000105612 Deoxyribonuclease II, lysosomal DNASE2BENSG00000137976 Deoxyribonuclease II beta DPEP1 ENSG00000015413Dipeptidase 1 (renal) DPEP2 ENSG00000167261 Dipeptidase 2 DPEP3ENSG00000141096 Dipeptidase 3 DPF3 ENSG00000205683 D4, zinc and doublePHD fingers, family 3 DPP4 ENSG00000197635 Dipeptidyl-peptidase 4 DPP7ENSG00000176978 Dipeptidyl-peptidase 7 DPT ENSG00000143196 DermatopontinDRAXIN ENSG00000162490 Dorsal inhibitory axon guidance protein DSEENSG00000111817 Dermatan sulfate epimerase DSG2 ENSG00000046604Desmoglein 2 DSPP ENSG00000152591 Dentin sialophosphoprotein DSTENSG00000151914 Dystonin DUOX1 ENSG00000137857 Dual oxidase 1 DYNLT3ENSG00000165169 Dynein, light chain, Tctex-type 3 E2F5 ENSG00000133740E2F transcription factor 5, p130-binding EBAG9 ENSG00000147654 Estrogenreceptor binding site associated, antigen, 9 EBI3 ENSG00000105246Epstein-Barr virus induced 3 ECHDC1 ENSG00000093144 Ethylmalonyl-CoAdecarboxylase 1 ECM1 ENSG00000143369 Extracellular matrix protein 1 ECM2ENSG00000106823 Extracellular matrix protein 2, female organ andadipocyte specific ECSIT ENSG00000130159 ECSIT signalling integratorEDDM3A ENSG00000181562 Epididymal protein 3A EDDM3B ENSG00000181552Epididymal protein 3B EDEM2 ENSG00000088298 ER degradation enhancer,mannosidase alpha-like 2 EDEM3 ENSG00000116406 ER degradation enhancer,mannosidase alpha-like 3 EDIL3 ENSG00000164176 EGF-like repeats anddiscoidin I-like domains 3 EDN1 ENSG00000078401 Endothelin 1 EDN2ENSG00000127129 Endothelin 2 EDN3 ENSG00000124205 Endothelin 3 EDNRBENSG00000136160 Endothelin receptor type B EFEMP1 ENSG00000115380 EGFcontaining fibulin-like extracellular matrix protein 1 EFEMP2ENSG00000172638 EGF containing fibulin-like extracellular matrix protein2 EFNA1 ENSG00000169242 Ephrin-Al EFNA2 ENSG00000099617 Ephrin-A2 EFNA4ENSG00000243364 Ephrin-A4 EGFL6 ENSG00000198759 EGF-like-domain,multiple 6 EGFL7 ENSG00000172889 EGF-like-domain, multiple 7 EGFL8ENSG00000241404 EGF-like-domain, multiple 8 EGFLAM ENSG00000164318EGF-like, fibronectin type III and laminin G domains EGFRENSG00000146648 Epidermal growth factor receptor EHBP1 ENSG00000115504EH domain binding protein 1 EHF ENSG00000135373 Ets homologous factorEHMT1 ENSG00000181090 Euchromatic histone-lysine N-methyltransferase 1EHMT2 ENSG00000204371 Euchromatic histone-lysine N-methyltransferase 2EIF2AK1 ENSG00000086232 Eukaryotic translation initiation factor 2-alphakinase 1 ELANE ENSG00000197561 Elastase, neutrophil expressed ELNENSG00000049540 Elastin ELP2 ENSG00000134759 Elongator acetyltransferasecomplex subunit 2 EL SPBP1 ENSG00000169393 Epididymal sperm bindingprotein 1 EMC1 ENSG00000127463 ER membrane protein complex subunit 1EMC10 ENSG00000161671 ER membrane protein complex subunit 10 EMC9ENSG00000100908 ER membrane protein complex subunit 9 EMCNENSG00000164035 Endomucin EMID1 ENSG00000186998 EMI domain containing 1EMILIN1 ENSG00000138080 Elastin microfibril interfacer 1 EMILIN2ENSG00000132205 Elastin microfibril interfacer 2 EMILIN3 ENSG00000183798Elastin microfibril interfacer 3 ENAM ENSG00000132464 Enamelin ENDOGENSG00000167136 Endonuclease G ENDOU ENSG00000111405 Endonuclease,polyU-specific ENHO ENSG00000168913 Energy homeostasis associated EN04ENSG00000188316 Enolase family member 4 ENPP6 ENSG00000164303Ectonucleotide pyrophosphatase/phosphodiesterase 6 ENPP7 ENSG00000182156Ectonucleotide pyrophosphatase/phosphodiesterase 7 ENTPD5ENSG00000187097 Ectonucleoside triphosphate diphosphohydrolase 5 ENTPD8ENSG00000188833 Ectonucleoside triphosphate diphosphohydrolase 8 EOGTENSG00000163378 EGF domain-specific O-linked N-acetylglucosamine(GlcNAc) transferase EPCAM ENSG00000119888 Epithelial cell adhesionmolecule EPDR1 ENSG00000086289 Ependymin related 1 EPGN ENSG00000182585Epithelial mitogen EPHA10 ENSG00000183317 EPH receptor A10 EPHA3ENSG00000044524 EPH receptor A3 EPHA4 ENSG00000116106 EPH receptor A4EPHA7 ENSG00000135333 EPH receptor A7 EPHA8 ENSG00000070886 EPH receptorA8 EPHB2 ENSG00000133216 EPH receptor B2 EPHB4 ENSG00000196411 EPHreceptor B4 EPHX3 ENSG00000105131 Epoxide hydrolase 3 EPOENSG00000130427 Erythropoietin EPPIN ENSG00000101448 Epididymalpeptidase inhibitor EPPIN-WFDC6 ENSG00000249139 EPPIN-WFDC6 readthroughEPS15 ENSG00000085832 Epidermal growth factor receptor pathway substrate15 EPS8L1 ENSG00000131037 EPS8-like 1 EPX ENSG00000121053 Eosinophilperoxidase EPYC ENSG00000083782 Epiphycan EQTN ENSG00000120160Equatorin, sperm acrosome associated ERAP1 ENSG00000164307 Endoplasmicreticulum aminopeptidase 1 ERAP2 ENSG00000164308 Endoplasmic reticulumaminopeptidase 2 ERBB3 ENSG00000065361 Erb-b2 receptor tyrosine kinase 3ERLIN1 ENSG00000107566 ER lipid raft associated 1 ERLIN2 ENSG00000147475ER lipid raft associated 2 ERNI ENSG00000178607 Endoplasmic reticulum tonucleus signaling 1 ERN2 ENSG00000134398 Endoplasmic reticulum tonucleus signaling 2 ERO1A ENSG00000197930 Endoplasmic reticulumoxidoreductase alpha ERO1B ENSG00000086619 Endoplasmic reticulumoxidoreductase beta ERP27 ENSG00000139055 Endoplasmic reticulum protein27 ERP29 ENSG00000089248 Endoplasmic reticulum protein 29 ERP44ENSG00000023318 Endoplasmic reticulum protein 44 ERV3-1 ENSG00000213462Endogenous retrovirus group 3, member 1 ESM1 ENSG00000164283 Endothelialcell-specific molecule 1 ESRP1 ENSG00000104413 Epithelial splicingregulatory protein 1 EXOG ENSG00000157036 Endo/exonuclease (5′-3′),endonuclease G-like EXTL1 ENSG00000158008 Exostosin-likeglycosyltransferase 1 EXTL2 ENSG00000162694 Exostosin-likeglycosyltransferase 2 F10 ENSG00000126218 Coagulation factor X F11ENSG00000088926 Coagulation factor XI F12 ENSG00000131187 Coagulationfactor XII (Hageman factor) F13B ENSG00000143278 Coagulation factorXIII, B polypeptide F2 ENSG00000180210 Coagulation factor II (thrombin)F2R ENSG00000181104 Coagulation factor II (thrombin) receptor F2RL3ENSG00000127533 Coagulation factor II (thrombin) receptor-like 3 F5ENSG00000198734 Coagulation factor V (proaccelerin, labile factor) F7ENSG00000057593 Coagulation factor VII (serum prothrombin conversionaccelerator) F8 ENSG00000185010 Coagulation factor VIII, procoagulantcomponent F9 ENSG00000101981 Coagulation factor IX FABP6 ENSG00000170231Fatty acid binding protein 6, ileal FAM107B ENSG00000065809 Family withsequence similarity 107, member B FAM131A ENSG00000175182 Family withsequence similarity 131, member A FAM132A ENSG00000184163 Family withsequence similarity 132, member A FAM132B ENSG00000178752 Family withsequence similarity 132, member B FAM150A ENSG00000196711 Family withsequence similarity 150, member A FAM150B ENSG00000189292 Family withsequence similarity 150, member B FAM171A1 ENSG00000148468 Family withsequence similarity 171, member Al FAM171B ENSG00000144369 Family withsequence similarity 171, member B FAM172A ENSG00000113391 Family withsequence similarity 172, member A FAM175A ENSG00000163322 Family withsequence similarity 175, member A FAM177A1 ENSG00000151327 Family withsequence similarity 177, member Al FAM179B ENSG00000198718 Family withsequence similarity 179, member B FAM180A ENSG00000189320 Family withsequence similarity 180, member A FAM189A1 ENSG00000104059 Family withsequence similarity 189, member Al FAM198A ENSG00000144649 Family withsequence similarity 198, member A FAM19A1 ENSG00000183662 Family withsequence similarity 19 (chemokine (C-C motif)-like), member A1 FAM19A2ENSG00000198673 Family with sequence similarity 19 (chemokine (C-Cmotif)-like), member A2 FAM19A3 ENSG00000184599 Family with sequencesimilarity 19 (chemokine (C-C motif)-like), member A3 FAM19A4ENSG00000163377 Family with sequence similarity 19 (chemokine (C-Cmotif)-like), member A4 FAM19A5 ENSG00000219438 Family with sequencesimilarity 19 (chemokine (C-C motif)-like), member A5 FAM20AENSG00000108950 Family with sequence similarity 20, member A FAM20CENSG00000177706 Family with sequence similarity 20, member C FAM213AENSG00000122378 Family with sequence similarity 213, member A FAM26DENSG00000164451 Family with sequence similarity 26, member D FAM46BENSG00000158246 Family with sequence similarity 46, member B FAM57AENSG00000167695 Family with sequence similarity 57, member A FAM78AENSG00000126882 Family with sequence similarity 78, member A FAM96AENSG00000166797 Family with sequence similarity 96, member A FAM9BENSG00000177138 Family with sequence similarity 9, member B FAPENSG00000078098 Fibroblast activation protein, alpha FAS ENSG00000026103Fas cell surface death receptor FAT1 ENSG00000083857 FAT atypicalcadherin 1 FBLN1 ENSG00000077942 Fibulin 1 FBLN2 ENSG00000163520 Fibulin2 FBLN5 ENSG00000140092 Fibulin 5 FBLN7 ENSG00000144152 Fibulin 7 FBN1ENSG00000166147 Fibrillin 1 FBN2 ENSG00000138829 Fibrillin 2 FBN3ENSG00000142449 Fibrillin 3 FBXW7 ENSG00000109670 F-box and WD repeatdomain containing 7, E3 ubiquitin protein ligase FCAR ENSG00000186431 Fcfragment of IgA receptor FCGBP ENSG00000275395 Fc fragment of IgGbinding protein FCGR1B ENSG00000198019 Fc fragment of IgG, high affinitylb, receptor (CD64) FCGR3A ENSG00000203747 Fc fragment of IgG, lowaffinity Ma, receptor (CD16a) FCGRT ENSG00000104870 Fc fragment of IgG,receptor, transporter, alpha FCMR ENSG00000162894 Fc fragment of IgMreceptor FCN1 ENSG00000085265 Ficolin (collagen/fibrinogen domaincontaining) 1 FCN2 ENSG00000160339 Ficolin (collagen/fibrinogen domaincontaining lectin) 2 FCN3 ENSG00000142748 Ficolin (collagen/fibrinogendomain containing) 3 FCRL1 ENSG00000163534 Fc receptor-like 1 FCRL3ENSG00000160856 Fc receptor-like 3 FCRL5 ENSG00000143297 Fcreceptor-like 5 FCRLA ENSG00000132185 Fc receptor-like A FCRLBENSG00000162746 Fc receptor-like B FDCSP ENSG00000181617 Folliculardendritic cell secreted protein FETUB ENSG00000090512 Fetuin B FGAENSG00000171560 Fibrinogen alpha chain FGB ENSG00000171564 Fibrinogenbeta chain FGF10 ENSG00000070193 Fibroblast growth factor 10 FGF17ENSG00000158815 Fibroblast growth factor 17 FGF18 ENSG00000156427Fibroblast growth factor 18 FGF19 ENSG00000162344 Fibroblast growthfactor 19 FGF21 ENSG00000105550 Fibroblast growth factor 21 FGF22ENSG00000070388 Fibroblast growth factor 22 FGF23 ENSG00000118972Fibroblast growth factor 23 FGF3 ENSG00000186895 Fibroblast growthfactor 3 FGF4 ENSG00000075388 Fibroblast growth factor 4 FGF5ENSG00000138675 Fibroblast growth factor 5 FGF7 ENSG00000140285Fibroblast growth factor 7 FGF8 ENSG00000107831 Fibroblast growth factor8 (androgen-induced) FGFBP1 ENSG00000137440 Fibroblast growth factorbinding protein 1 FGFBP2 ENSG00000137441 Fibroblast growth factorbinding protein 2 FGFBP3 ENSG00000174721 Fibroblast growth factorbinding protein 3 FGFR1 ENSG00000077782 Fibroblast growth factorreceptor 1 FGFR2 ENSG00000066468 Fibroblast growth factor receptor 2FGFR3 ENSG00000068078 Fibroblast growth factor receptor 3 FGFR4ENSG00000160867 Fibroblast growth factor receptor 4 FGFRL1ENSG00000127418 Fibroblast growth factor receptor-like 1 FGGENSG00000171557 Fibrinogen gamma chain FGL1 ENSG00000104760Fibrinogen-like 1 FGL2 ENSG00000127951 Fibrinogen-like 2 FHL1ENSG00000022267 Four and a half LIM domains 1 FHOD3 ENSG00000134775Formin homology 2 domain containing 3 FIBIN ENSG00000176971 Fin budinitiation factor homolog (zebrafish) FICD ENSG00000198855 FIC domaincontaining FIGF ENSG00000165197 C-fos induced growth factor (vascularendothelial growth factor D) FJX1 ENSG00000179431 Four jointed box 1FKBP10 ENSG00000141756 FK506 binding protein 10, 65 kDa FKBP11ENSG00000134285 FK506 binding protein 11, 19 kDa FKBP14 ENSG00000106080FK506 binding protein 14, 22 kDa FKBP2 ENSG00000173486 FK506 bindingprotein 2, 13 kDa FKBP7 ENSG00000079150 FK506 binding protein 7 FKBP9ENSG00000122642 FK506 binding protein 9, 63 kDa FLT1 ENSG00000102755Fms-related tyrosine kinase 1 FLT4 ENSG00000037280 Fms-related tyrosinekinase 4 FM01 ENSG00000010932 Flavin containing monooxygenase 1 FM02ENSG00000094963 Flavin containing monooxygenase 2 (non-functional) FM03ENSG00000007933 Flavin containing monooxygenase 3 FM05 ENSG00000131781Flavin containing monooxygenase 5 FMOD ENSG00000122176 Fibromodulin FN1ENSG00000115414 Fibronectin 1 FNDC1 ENSG00000164694 Fibronectin type IIIdomain containing 1 FNDC7 ENSG00000143107 Fibronectin type III domaincontaining 7 FOCAD ENSG00000188352 Focadhesin FOLR2 ENSG00000165457Folate receptor 2 (fetal) FOLR3 ENSG00000110203 Folate receptor 3(gamma) FOXRED2 ENSG00000100350 FAD-dependent oxidoreductase domaincontaining 2 FP325331.1 ENSG00000278881 Uncharacterized proteinUNQ6126/PR020091 FPGS ENSG00000136877 Folylpolyglutamate synthase FRAS1ENSG00000138759 Fraser extracellular matrix complex subunit 1 FREM1ENSG00000164946 FRAS1 related extracellular matrix 1 FREM3ENSG00000183090 FRAS1 related extracellular matrix 3 FRMPD2ENSG00000170324 FERM and PDZ domain containing 2 FRZB ENSG00000162998Frizzled-related protein FSHB ENSG00000131808 Follicle stimulatinghormone, beta polypeptide FSHR ENSG00000170820 Follicle stimulatinghormone receptor FST ENSG00000134363 Follistatin FSTL1 ENSG00000163430Follistatin-like 1 FSTL3 ENSG00000070404 Follistatin-like 3 (secretedglycoprotein) FSTL4 ENSG00000053108 Follistatin-like 4 FSTL5ENSG00000168843 Follistatin-like 5 FTCDNL1 ENSG00000226124Formiminotransferase cyclodeaminase N-terminal like FUCA1ENSG00000179163 Fucosidase, alpha-L- 1, tissue FUCA2 ENSG00000001036Fucosidase, alpha-L- 2, plasma FURIN ENSG00000140564 Furin (paired basicamino acid cleaving enzyme) FUT10 ENSG00000172728 Fucosyltransferase 10(alpha (1,3) fucosyltransferase) FUT11 ENSG00000196968Fucosyltransferase 11 (alpha (1,3) fucosyltransferase) FXNENSG00000165060 Frataxin FXR1 ENSG00000114416 Fragile X mentalretardation, autosomal homolog 1 FXYD3 ENSG00000089356 FXYD domaincontaining ion transport regulator 3 GABBR1 ENSG00000204681Gamma-aminobutyric acid (GABA) B receptor, 1 GABRA1 ENSG00000022355Gamma-aminobutyric acid (GABA) A receptor, alpha 1 GABRA2ENSG00000151834 Gamma-aminobutyric acid (GABA) A receptor, alpha 2GABRA5 ENSG00000186297 Gamma-aminobutyric acid (GABA) A receptor, alpha5 GABRG3 ENSG00000182256 Gamma-aminobutyric acid (GABA) A receptor,gamma 3 GABRP ENSG00000094755 Gamma-aminobutyric acid (GABA) A receptor,pi GAL ENSG00000069482 Galanin/GMAP prepropeptide GAL3ST1ENSG00000128242 Galactose-3-O-sulfotransferase 1 GAL3ST2 ENSG00000154252Galactose-3-O-sulfotransferase 2 GAL3ST3 ENSG00000175229Galactose-3-O-sulfotransferase 3 GALC ENSG00000054983Galactosylceramidase GALNS ENSG00000141012 Galactosamine(N-acetyl)-6-sulfatase GALNT10 ENSG00000164574 PolypeptideN-acetylgalactosaminyltransferase 10 GALNT12 ENSG00000119514 PolypeptideN-acetylgalactosaminyltransferase 12 GALNT15 ENSG00000131386 PolypeptideN-acetylgalactosaminyltransferase 15 GALNT2 ENSG00000143641 PolypeptideN-acetylgalactosaminyltransferase 2 GALNT6 ENSG00000139629 PolypeptideN-acetylgalactosaminyltransferase 6 GALNT8 ENSG00000130035 PolypeptideN-acetylgalactosaminyltransferase 8 GALNTL6 ENSG00000174473 PolypeptideN-acetylgalactosaminyltransferase-like 6 GALP ENSG00000197487Galanin-like peptide GANAB ENSG00000089597 Glucosidase, alpha; neutralAB GARS ENSG00000106105 Glycyl-tRNA synthetase GAS1 ENSG00000180447Growth arrest-specific 1 GAS6 ENSG00000183087 Growth arrest-specific 6GAST ENSG00000184502 Gastrin GBA ENSG00000177628 Glucosidase, beta, acidGBGT1 ENSG00000148288 Globosidealpha-1,3-N-acetylgalactosaminyltransferase 1 GC ENSG00000145321Group-specific component (vitamin D binding protein) GCG ENSG00000115263Glucagon GCGR ENSG00000215644 Glucagon receptor GCNT7 ENSG00000124091Glucosaminyl (N-acetyl) transferase family member 7 GCSH ENSG00000140905Glycine cleavage system protein H (aminomethyl carrier) GDF1ENSG00000130283 Growth differentiation factor 1 GDF10 ENSG00000266524Growth differentiation factor 10 GDF11 ENSG00000135414 Growthdifferentiation factor 11 GDF15 ENSG00000130513 Growth differentiationfactor 15 GDF2 ENSG00000263761 Growth differentiation factor 2 GDF3ENSG00000184344 Growth differentiation factor 3 GDF5 ENSG00000125965Growth differentiation factor 5 GDF6 ENSG00000156466 Growthdifferentiation factor 6 GDF7 ENSG00000143869 Growth differentiationfactor 7 GDF9 ENSG00000164404 Growth differentiation factor 9 GDNFENSG00000168621 Glial cell derived neurotrophic factor GFOD2ENSG00000141098 Glucose-fructose oxidoreductase domain containing 2GFPT2 ENSG00000131459 Glutamine-fructose-6-phosphate transaminase 2GFRA2 ENSG00000168546 GDNF family receptor alpha 2 GFRA4 ENSG00000125861GDNF family receptor alpha 4 GGA2 ENSG00000103365 Golgi-associated,gamma adaptin ear containing, ARF binding protein 2 GGH ENSG00000137563Gamma-glutamyl hydrolase (conjugase, folylpolygammaglutamyl hydrolase)GGT1 ENSG00000100031 Gamma-glutamyltransferase 1 GGT5 ENSG00000099998Gamma-glutamyltransferase 5 GH1 ENSG00000259384 Growth hormone 1 GH2ENSG00000136487 Growth hormone 2 GHDC ENSG00000167925 GH3 domaincontaining GHRH ENSG00000118702 Growth hormone releasing hormone GHRHRENSG00000106128 Growth hormone releasing hormone receptor GHRLENSG00000157017 Ghrelin/obestatin prepropeptide GIF ENSG00000134812Gastric intrinsic factor (vitamin B synthesis) GIP ENSG00000159224Gastric inhibitory polypeptide GKN1 ENSG00000169605 Gastrokine 1 GKN2ENSG00000183607 Gastrokine 2 GLA ENSG00000102393 Galactosidase, alphaGLB1 ENSG00000170266 Galactosidase, beta 1 GLB1L ENSG00000163521Galactosidase, beta 1-like GLB1L2 ENSG00000149328 Galactosidase, beta1-like 2 GLCE ENSG00000138604 Glucuronic acid epimerase GLG1ENSG00000090863 Golgi glycoprotein 1 GLIPR1 ENSG00000139278 GLIpathogenesis-related 1 GLIPR1L1 ENSG00000173401 GLI pathogenesis-related1 like 1 GLIS3 ENSG00000107249 GUS family zinc finger 3 GLMPENSG00000198715 Glycosylated lysosomal membrane protein GLRBENSG00000109738 Glycine receptor, beta GLS ENSG00000115419 GlutaminaseGLT6D1 ENSG00000204007 Glycosyltransferase 6 domain containing 1 GLTPD2ENSG00000182327 Glycolipid transfer protein domain containing 2 GLUD1ENSG00000148672 Glutamate dehydrogenase 1 GM2A ENSG00000196743 GM2ganglioside activator GML ENSG00000104499 Glycosylphosphatidylinositolanchored molecule like GNAS ENSG00000087460 GNAS complex locus GNLYENSG00000115523 Granulysin GNPTG ENSG00000090581N-acetylglucosamine-1-phosphate transferase, gamma subunit GNRH1ENSG00000147437 Gonadotropin-releasing hormone 1 (luteinizing-releasinghormone) GNRH2 ENSG00000125787 Gonadotropin-releasing hormone 2 GNSENSG00000135677 Glucosamine (N-acetyl)-6-sulfatase GOLM1 ENSG00000135052Golgi membrane protein 1 GORAB ENSG00000120370 Golgin, RAB6-interactingGOT2 ENSG00000125166 Glutamic-oxaloacetic transaminase 2, mitochondrialGP2 ENSG00000169347 Glycoprotein 2 (zymogen granule membrane) GP6ENSG00000088053 Glycoprotein VI (platelet) GPC2 ENSG00000213420 Glypican2 GPC5 ENSG00000179399 Glypican 5 GPC6 ENSG00000183098 Glypican 6 GPD2ENSG00000115159 Glycerol-3-phosphate dehydrogenase 2 (mitochondrial)GPER1 ENSG00000164850 G protein-coupled estrogen receptor 1 GPHA2ENSG00000149735 Glycoprotein hormone alpha 2 GPHB5 ENSG00000179600Glycoprotein hormone beta 5 GPIHBP1 ENSG00000277494Glycosylphosphatidylinositol anchored high density lipoprotein bindingprotein 1 GPLD1 ENSG00000112293 Glycosylphosphatidylinositol specificphospholipase D1 GPNMB ENSG00000136235 Glycoprotein (transmembrane) nmbGPR162 ENSG00000250510 G protein-coupled receptor 162 GPX3ENSG00000211445 Glutathione peroxidase 3 GPX4 ENSG00000167468Glutathione peroxidase 4 GPX5 ENSG00000224586 Glutathione peroxidase 5GPX6 ENSG00000198704 Glutathione peroxidase 6 GPX7 ENSG00000116157Glutathione peroxidase 7 GREM1 ENSG00000166923 Gremlin 1, DAN family BMPantagonist GREM2 ENSG00000180875 Gremlin 2, DAN family BMP antagonistGRHL3 ENSG00000158055 Grainyhead-like transcription factor 3 GRIA2ENSG00000120251 Glutamate receptor, ionotropic, AMPA 2 GRIA3ENSG00000125675 Glutamate receptor, ionotropic, AMPA 3 GRIA4ENSG00000152578 Glutamate receptor, ionotropic, AMPA 4 GRIK2ENSG00000164418 Glutamate receptor, ionotropic, kainate 2 GRIN2BENSG00000273079 Glutamate receptor, ionotropic, N-methyl D-aspartate 2BGRM2 ENSG00000164082 Glutamate receptor, metabotropic 2 GRM3ENSG00000198822 Glutamate receptor, metabotropic 3 GRM5 ENSG00000168959Glutamate receptor, metabotropic 5 GRN ENSG00000030582 Granulin GRPENSG00000134443 Gastrin-releasing peptide GSG1 ENSG00000111305 Germ cellassociated 1 GSN ENSG00000148180 Gelsolin GTDC1 ENSG00000121964Glycosyltransferase-like domain containing 1 GTPBP10 ENSG00000105793GTP-binding protein 10 (putative) GUCA2A ENSG00000197273 Guanylatecyclase activator 2A (guanylin) GUCA2B ENSG00000044012 Guanylate cyclaseactivator 2B (uroguanylin) GUSB ENSG00000169919 Glucuronidase, betaGVQW1 ENSG00000241043 GVQW motif containing 1 GXYLT1 ENSG00000151233Glucoside xylosyltransferase 1 GXYLT2 ENSG00000172986 Glucosidexylosyltransferase 2 GYLTL1B ENSG00000165905 Glycosyltransferase-like 1BGYPB ENSG00000250361 Glycophorin B (MNS blood group) GZMAENSG00000145649 Granzyme A (granzyme 1, cytotoxicT-lymphocyte-associated serine esterase 3) GZMB ENSG00000100453 GranzymeB (granzyme 2, cytotoxic T-lymphocyte-associated serine esterase 1) GZMHENSG00000100450 Granzyme H (cathepsin G-like 2, protein h-CCPX) GZMKENSG00000113088 Granzyme K (granzyme 3; tryptase II) GZMIMENSG00000197540 Granzyme M (lymphocyte met-ase 1) H6PD ENSG00000049239Hexose-6-phosphate dehydrogenase (glucose 1-dehydrogenase) HABP2ENSG00000148702 Hyaluronan binding protein 2 HADHB ENSG00000138029Hydroxyacyl-CoA dehydrogenase/3-ketoacyl-CoA thiolase/enoyl-CoAhydratase (trifunctional protein), beta subunit HAMP ENSG00000105697Hepcidin antimicrobial peptide HAPLN1 ENSG00000145681 Hyaluronan andproteoglycan link protein 1 HAPLN2 ENSG00000132702 Hyaluronan andproteoglycan link protein 2 HAPLN3 ENSG00000140511 Hyaluronan andproteoglycan link protein 3 HAPLN4 ENSG00000187664 Hyaluronan andproteoglycan link protein 4 HARS2 ENSG00000112855 Histidyl-tRNAsynthetase 2, mitochondrial HAVCR1 ENSG00000113249 Hepatitis A viruscellular receptor 1 HCCS ENSG00000004961 Holocytochrome c synthase HCRTENSG00000161610 Hypocretin (orexin) neuropeptide precursor HEATR5AENSG00000129493 HEAT repeat containing 5A HEPH ENSG00000089472Hephaestin HEXA ENSG00000213614 Hexosaminidase A (alpha polypeptide)HEXB ENSG00000049860 Hexosaminidase B (beta polypeptide) HFE2ENSG00000168509 Hemochromatosis type 2 (juvenile) HGF ENSG00000019991Hepatocyte growth factor (hepapoietin A; scatter factor) HGFACENSG00000109758 HGF activator HHIP ENSG00000164161 Hedgehog interactingprotein HHIPL1 ENSG00000182218 HHIP-like 1 HHIPL2 ENSG00000143512HHIP-like 2 HHLA1 ENSG00000132297 HERV-H LTR-associating 1 HHLA2ENSG00000114455 HERV-H LTR-associating 2 HIBADH ENSG000001060493-hydroxyisobutyrate dehydrogenase HINT2 ENSG00000137133 Histidine triadnucleotide binding protein 2 HLA-A ENSG00000206503 Majorhistocompatibility complex, class I, A HLA-C ENSG00000204525 Majorhistocompatibility complex, class I, C HLA-DOA ENSG00000204252 Majorhistocompatibility complex, class II, DO alpha HLA-DPA1 ENSG00000231389Major histocompatibility complex, class II, DP alpha 1 HLA-DQA1ENSG00000196735 Major histocompatibility complex, class II, DQ alpha 1HLA-DQB1 ENSG00000179344 Major histocompatibility complex, class II, DQbeta 1 HLA-DQB2 ENSG00000232629 Major histocompatibility complex, classII, DQ beta 2 HMCN1 ENSG00000143341 Hemicentin 1 HMCN2 ENSG00000148357Hemicentin 2 HMGCL ENSG00000117305 3-hydroxymethy1-3-methylglutaryl-CoAlyase HMHAl ENSG00000180448 Histocompatibility (minor) HA-1 HMSDENSG00000221887 Histocompatibility (minor) serpin domain containing HPENSG00000257017 Haptoglobin HPR ENSG00000261701 Haptoglobin-relatedprotein HPSE ENSG00000173083 Heparanase HPSE2 ENSG00000172987 Heparanase2 (inactive) HPX ENSG00000110169 Hemopexin HRC ENSG00000130528 Histidinerich calcium binding protein HRG ENSG00000113905 Histidine-richglycoprotein HRSP12 ENSG00000132541 Heat-responsive protein 12 HS25T1ENSG00000153936 Heparan sulfate 2-O-sulfotransferase 1 HS3ST1ENSG00000002587 Heparan sulfate (glucosamine) 3-O-sulfotransferase 1HS65T1 ENSG00000136720 Heparan sulfate 6-O-sulfotransferase 1 HS65T3ENSG00000185352 Heparan sulfate 6-O-sulfotransferase 3 HSD11B1LENSG00000167733 Hydroxysteroid (11-beta) dehydrogenase 1-like HSD17B11ENSG00000198189 Hydroxysteroid (17-beta) dehydrogenase 11 HSD17B7ENSG00000132196 Hydroxysteroid (17-beta) dehydrogenase 7 HSP90B1ENSG00000166598 Heat shock protein 90 kDa beta (Grp94), member 1 HSPA13ENSG00000155304 Heat shock protein 70 kDa family, member 13 HSPA5ENSG00000044574 Heat shock 70 kDa protein 5 (glucose-regulated protein,78 kDa) HSPG2 ENSG00000142798 Heparan sulfate proteoglycan 2 HTATIP2ENSG00000109854 HIV-1 Tat interactive protein 2, 30 kDa HTN1ENSG00000126550 Histatin 1 HTN3 ENSG00000205649 Histatin 3 HTRA1ENSG00000166033 HtrA serine peptidase 1 HTRA3 ENSG00000170801 HtrAserine peptidase 3 HTRA4 ENSG00000169495 HtrA serine peptidase 4 HYAL1ENSG00000114378 Hyaluronoglucosaminidase 1 HYAL2 ENSG00000068001Hyaluronoglucosaminidase 2 HYAL3 ENSG00000186792Hyaluronoglucosaminidase 3 HYOU1 ENSG00000149428 Hypoxia up-regulated 1IAPP ENSG00000121351 Islet amyloid polypeptide TBSP ENSG00000029559Integrin-binding sialoprotein ICAM1 ENSG00000090339 Intercellularadhesion molecule 1 ICAM2 ENSG00000108622 Intercellular adhesionmolecule 2 ICAM4 ENSG00000105371 Intercellular adhesion molecule 4(Landsteiner-Wiener blood group) ID1 ENSG00000125968 Inhibitor of DNAbinding 1, dominant negative helix-loop-helix protein IDEENSG00000119912 Insulin-degrading enzyme IDNK ENSG00000148057 IdnK,gluconokinase homolog (E. coli) IDS ENSG00000010404 Iduronate2-sulfatase IDUA ENSG00000127415 Iduronidase, alpha-L- IFI27L2ENSG00000119632 Interferon, alpha-inducible protein 27-like 2 IFI30ENSG00000216490 Interferon, gamma-inducible protein 30 IFNA1ENSG00000197919 Interferon, alpha 1 IFNA10 ENSG00000186803 Interferon,alpha 10 IFNA13 ENSG00000233816 Interferon, alpha 13 IFNA14ENSG00000228083 Interferon, alpha 14 IFNA16 ENSG00000147885 Interferon,alpha 16 IFNA17 ENSG00000234829 Interferon, alpha 17 IFNA2ENSG00000188379 Interferon, alpha 2 IFNA21 ENSG00000137080 Interferon,alpha 21 IFNA4 ENSG00000236637 Interferon, alpha 4 IFNA5 ENSG00000147873Interferon, alpha 5 IFNA6 ENSG00000120235 Interferon, alpha 6 IFNA7ENSG00000214042 Interferon, alpha 7 IFNA8 ENSG00000120242 Interferon,alpha 8 IFNAR1 ENSG00000142166 Interferon (alpha, beta and omega)receptor 1 IFNB1 ENSG00000171855 Interferon, beta 1, fibroblast IFNEENSG00000184995 Interferon, epsilon IFNG ENSG00000111537 Interferon,gamma IFNGR1 ENSG00000027697 Interferon gamma receptor 1 IFNL1ENSG00000182393 Interferon, lambda 1 IFNL2 ENSG00000183709 Interferon,lambda 2 IFNL3 ENSG00000197110 Interferon, lambda 3 IFNLR1ENSG00000185436 Interferon, lambda receptor 1 IFNW1 ENSG00000177047Interferon, omega 1 IGF1 ENSG00000017427 Insulin-like growth factor 1(somatomedin C) IGF2 ENSG00000167244 Insulin-like growth factor 2 IGFALSENSG00000099769 Insulin-like growth factor binding protein, acid labilesubunit IGFBP1 ENSG00000146678 Insulin-like growth factor bindingprotein 1 IGFBP2 ENSG00000115457 Insulin-like growth factor bindingprotein 2, 36 kDa IGFBP3 ENSG00000146674 Insulin-like growth factorbinding protein 3 IGFBP4 ENSG00000141753 Insulin-like growth factorbinding protein 4 IGFBP5 ENSG00000115461 Insulin-like growth factorbinding protein 5 IGFBP6 ENSG00000167779 Insulin-like growth factorbinding protein 6 IGFBP7 ENSG00000163453 Insulin-like growth factorbinding protein 7 IGFBPL1 ENSG00000137142 Insulin-like growth factorbinding protein-like 1 IGFL1 ENSG00000188293 IGF-like family member 1IGFL2 ENSG00000204866 IGF-like family member 2 IGFL3 ENSG00000188624IGF-like family member 3 IGFLR1 ENSG00000126246 IGF-like family receptor1 IGIP ENSG00000182700 IgA-inducing protein IGLON5 ENSG00000142549 IgLONfamily member 5 IGSF1 ENSG00000147255 Immunoglobulin superfamily, member1 IGSF10 ENSG00000152580 Immunoglobulin superfamily, member 10 IGSF11ENSG00000144847 Immunoglobulin superfamily, member 11 IGSF21ENSG00000117154 Immunoglobin superfamily, member 21 IGSF8ENSG00000162729 Immunoglobulin superfamily, member 8 IGSF9ENSG00000085552 Immunoglobulin superfamily, member 9 IHH ENSG00000163501Indian hedgehog IL10 ENSG00000136634 Interleukin 10 IL11 ENSG00000095752Interleukin 11 IL11RA ENSG00000137070 Interleukin 11 receptor, alphaIL12B ENSG00000113302 Interleukin 12B IL12RB1 ENSG00000096996Interleukin 12 receptor, beta 1 IL12RB2 ENSG00000081985 Interleukin 12receptor, beta 2 IL13 ENSG00000169194 Interleukin 13 IL13RA1ENSG00000131724 Interleukin 13 receptor, alpha 1 IL15RA ENSG00000134470Interleukin 15 receptor, alpha IL17A ENSG00000112115 Interleukin 17AIL17B ENSG00000127743 Interleukin 17B IL17C ENSG00000124391 Interleukin17C IL17D ENSG00000172458 Interleukin 17D IL17F ENSG00000112116Interleukin 17F IL17RA ENSG00000177663 Interleukin 17 receptor A IL17RCENSG00000163702 Interleukin 17 receptor C IL17RE ENSG00000163701Interleukin 17 receptor E IL18BP ENSG00000137496 Interleukin 18 bindingprotein IL18R1 ENSG00000115604 Interleukin 18 receptor 1 IL18RAPENSG00000115607 Interleukin 18 receptor accessory protein IL19ENSG00000142224 Interleukin 19 IL1R1 ENSG00000115594 Interleukin 1receptor, type I IL1R2 ENSG00000115590 Interleukin 1 receptor, type IIIL1RAP ENSG00000196083 Interleukin 1 receptor accessory protein IL1RL1ENSG00000115602 Interleukin 1 receptor-like 1 IL1RL2 ENSG00000115598Interleukin 1 receptor-like 2 IL1RN ENSG00000136689 Interleukin 1receptor antagonist IL2 ENSG00000109471 Interleukin 2 IL20ENSG00000162891 Interleukin 20 IL20RA ENSG00000016402 Interleukin 20receptor, alpha IL21 ENSG00000138684 Interleukin 21 IL22 ENSG00000127318Interleukin 22 IL22RA2 ENSG00000164485 Interleukin 22 receptor, alpha 2IL23A ENSG00000110944 Interleukin 23, alpha subunit p19 IL24ENSG00000162892 Interleukin 24 IL25 ENSG00000166090 Interleukin 25 IL26ENSG00000111536 Interleukin 26 IL27 ENSG00000197272 Interleukin 27 IL2RBENSG00000100385 Interleukin 2 receptor, beta IL3 ENSG00000164399Interleukin 3 IL31 ENSG00000204671 Interleukin 31 IL31RA ENSG00000164509Interleukin 31 receptor A IL32 ENSG00000008517 Interleukin 32 IL34ENSG00000157368 Interleukin 34 IL3RA ENSG00000185291 Interleukin 3receptor, alpha (low affinity) IL4 ENSG00000113520 Interleukin 4 IL4I1ENSG00000104951 Interleukin 4 induced 1 IL4R ENSG00000077238 Interleukin4 receptor IL5 ENSG00000113525 Interleukin 5 IL5RA ENSG00000091181Interleukin 5 receptor, alpha IL6 ENSG00000136244 Interleukin 6 IL6RENSG00000160712 Interleukin 6 receptor IL6ST ENSG00000134352 Interleukin6 signal transducer IL7 ENSG00000104432 Interleukin 7 IL7RENSG00000168685 Interleukin 7 receptor IL9 ENSG00000145839 Interleukin 9ILDR1 ENSG00000145103 Immunoglobulin-like domain containing receptor 1ILDR2 ENSG00000143195 Immunoglobulin-like domain containing receptor 2IMP4 ENSG00000136718 IMP4, U3 small nucleolar ribonucleoprotein IMPG1ENSG00000112706 Interphotoreceptor matrix proteoglycan 1 INHAENSG00000123999 Inhibin, alpha INHBA ENSG00000122641 Inhibin, beta AINHBB ENSG00000163083 Inhibin, beta B INHBC ENSG00000175189 Inhibin,beta C INHBE ENSG00000139269 Inhibin, beta E INPP5A ENSG00000068383Inositol polyphosphate-5-phosphatase A INS ENSG00000254647 InsulinINS-IGF2 ENSG00000129965 INS-IGF2 readthrough INSL3 ENSG00000248099Insulin-like 3 (Leydig cell) INSL4 ENSG00000120211 Insulin-like 4(placenta) INSL5 ENSG00000172410 Insulin-like 5 INSL6 ENSG00000120210Insulin-like 6 INTS3 ENSG00000143624 Integrator complex subunit 3 IP011ENSG00000086200 Importin 11 IP09 ENSG00000198700 Importin 9 IQCF6ENSG00000214686 IQ motif containing F6 IRAK3 ENSG00000090376Interleukin-1 receptor-associated kinase 3 IRS4 ENSG00000133124 Insulinreceptor substrate 4 ISLR ENSG00000129009 Immunoglobulin superfamilycontaining leucine-rich repeat ISLR2 ENSG00000167178 Immunoglobulinsuperfamily containing leucine-rich repeat 2 ISM1 ENSG00000101230Isthmin 1, angiogenesis inhibitor ISM2 ENSG00000100593 Isthmin 2 ITGA4ENSG00000115232 Integrin, alpha 4 (antigen CD49D, alpha 4 subunit ofVLA-4 receptor) ITGA9 ENSG00000144668 Integrin, alpha 9 ITGALENSG00000005844 Integrin, alpha L (antigen CD11A (p180), lymphocytefunction- associated antigen 1; alpha polypeptide) ITGAX ENSG00000140678Integrin, alpha X (complement component 3 receptor 4 subunit) ITGB1ENSG00000150093 Integrin, beta 1 (fibronectin receptor, betapolypeptide, antigen CD29 includes MDF2, MSK12) ITGB2 ENSG00000160255Integrin, beta 2 (complement component 3 receptor 3 and 4 subunit) ITGB3ENSG00000259207 Integrin, beta 3 (platelet glycoprotein IIIa, antigenCD61) ITGB7 ENSG00000139626 Integrin, beta 7 ITGBL1 ENSG00000198542Integrin, beta-like 1 (with EGF-like repeat domains) ITIH1ENSG00000055957 Inter-alpha-trypsin inhibitor heavy chain 1 ITIH2ENSG00000151655 Inter-alpha-trypsin inhibitor heavy chain 2 ITIH3ENSG00000162267 Inter-alpha-trypsin inhibitor heavy chain 3 ITIH4ENSG00000055955 Inter-alpha-trypsin inhibitor heavy chain family, member4 ITIH5 ENSG00000123243 Inter-alpha-trypsin inhibitor heavy chainfamily, member 5 ITIH6 ENSG00000102313 Inter-alpha-trypsin inhibitorheavy chain family, member 6 ITLN1 ENSG00000179914 Intelectin 1(galactofuranose binding) ITLN2 ENSG00000158764 Intelectin 2 IZUMO1RENSG00000183560 IZUMO1 receptor, JUNO IZUMO4 ENSG00000099840 IZUMOfamily member 4 JCHAIN ENSG00000132465 Joining chain of multimeric IgAand IgM JMJD8 ENSG00000161999 Jumonji domain containing 8 JSRP1ENSG00000167476 Junctional sarcoplasmic reticulum protein 1 KANSL2ENSG00000139620 KAT8 regulatory NSL complex subunit 2 KAZALD1ENSG00000107821 Kazal-type serine peptidase inhibitor domain 1 KCNIP3ENSG00000115041 Kv channel interacting protein 3, calsenilin KCNK7ENSG00000173338 Potassium channel, two pore domain subfamily K, member 7KCNN4 ENSG00000104783 Potassium channel, calcium activatedintermediate/small conductance subfamily N alpha, member 4 KCNU1ENSG00000215262 Potassium channel, subfamily U, member 1 KCPENSG00000135253 Kielin/chordin-like protein KDELC1 ENSG00000134901 KDEL(Lys-Asp-Glu-Leu) containing 1 KDELC2 ENSG00000178202 KDEL(Lys-Asp-Glu-Leu) containing 2 KDM1A ENSG00000004487 Lysine (K)-specificdemethylase 1A KDM3B ENSG00000120733 Lysine (K)-specific demethylase 3BKDM6A ENSG00000147050 Lysine (K)-specific demethylase 6A KDM7AENSG00000006459 Lysine (K)-specific demethylase 7A KDSR ENSG000001195373-ketodihydrosphingosine reductase KERA ENSG00000139330 KeratocanKIAA0100 ENSG00000007202 KIAA0100 KIAA0319 ENSG00000137261 KIAA0319KIAA1324 ENSG00000116299 KIAA1324 KIFC2 ENSG00000167702 Kinesin familymember C2 KIR2DL4 ENSG00000189013 Killer cell immunoglobulin-likereceptor, two domains, long cytoplasmic tail, 4 KIR3DX1 ENSG00000104970Killer cell immunoglobulin-like receptor, three domains, X1 KIRREL2ENSG00000126259 Kin of IRRE like 2 (Drosophila) KISS1 ENSG00000170498KiSS-1 metastasis-suppressor KLHL11 ENSG00000178502 Kelch-like familymember 11 KLHL22 ENSG00000099910 Kelch-like family member 22 KLK1ENSG00000167748 Kallikrein 1 KLK10 ENSG00000129451 Kallikrein-relatedpeptidase 10 KLK11 ENSG00000167757 Kallikrein-related peptidase 11 KLK12ENSG00000186474 Kallikrein-related peptidase 12 KLK13 ENSG00000167759Kallikrein-related peptidase 13 KLK14 ENSG00000129437 Kallikrein-relatedpeptidase 14 KLK15 ENSG00000174562 Kallikrein-related peptidase 15 KLK2ENSG00000167751 Kallikrein-related peptidase 2 KLK3 ENSG00000142515Kallikrein-related peptidase 3 KLK4 ENSG00000167749 Kallikrein-relatedpeptidase 4 KLK5 ENSG00000167754 Kallikrein-related peptidase 5 KLK6ENSG00000167755 Kallikrein-related peptidase 6 KLK7 ENSG00000169035Kallikrein-related peptidase 7 KLK8 ENSG00000129455 Kallikrein-relatedpeptidase 8 KLK9 ENSG00000213022 Kallikrein-related peptidase 9 KLKB1ENSG00000164344 Kallikrein B, plasma (Fletcher factor) 1 KNDC1ENSG00000171798 Kinase non-catalytic C-lobe domain (KIND) containing 1KNG1 ENSG00000113889 Kininogen 1 KRBA2 ENSG00000184619 KRAB-A domaincontaining 2 KREMEN2 ENSG00000131650 Kringle containing transmembraneprotein 2 KRTDAP ENSG00000188508 Keratinocyte differentiation-associatedprotein L1CAM ENSG00000198910 L1 cell adhesion molecule L3MBTL2ENSG00000100395 L(3)mbt-like 2 (Drosophila) LA16c-380H5.3ENSG00000270168 LACE1 ENSG00000135537 Lactation elevated 1 LACRTENSG00000135413 Lacritin LACTB ENSG00000103642 Lactamase, beta LAG3ENSG00000089692 Lymphocyte-activation gene 3 LAIR2 ENSG00000167618Leukocyte-associated immunoglobulin-like receptor 2 LALBAENSG00000167531 Lactalbumin, alpha- LAMA1 ENSG00000101680 Laminin, alpha1 LAMA2 ENSG00000196569 Laminin, alpha 2 LAMA3 ENSG00000053747 Laminin,alpha 3 LAMA4 ENSG00000112769 Laminin, alpha 4 LAMA5 ENSG00000130702Laminin, alpha 5 LAMB1 ENSG00000091136 Laminin, beta 1 LAMB2ENSG00000172037 Laminin, beta 2 (laminin S) LAMB3 ENSG00000196878Laminin, beta 3 LAMB4 ENSG00000091128 Laminin, beta 4 LAMC1ENSG00000135862 Laminin, gamma 1 (formerly LAMB2) LAMC2 ENSG00000058085Laminin, gamma 2 LAMC3 ENSG00000050555 Laminin, gamma 3 LAMP3ENSG00000078081 Lysosomal-associated membrane protein 3 LATENSG00000213658 Linker for activation of T cells LAT2 ENSG00000086730Linker for activation of T cells family, member 2 LBP ENSG00000129988Lipopolysaccharide binding protein LCAT ENSG00000213398Lecithin-cholesterol acyltransferase LCN1 ENSG00000160349 Lipocalin 1LCN10 ENSG00000187922 Lipocalin 10 LCN12 ENSG00000184925 Lipocalin 12LCN15 ENSG00000177984 Lipocalin 15 LCN2 ENSG00000148346 Lipocalin 2 LCN6ENSG00000267206 Lipocalin 6 LCN8 ENSG00000204001 Lipocalin 8 LCN9ENSG00000148386 Lipocalin 9 LCORL ENSG00000178177 Ligand dependentnuclear receptor corepressor-like LDLR ENSG00000130164 Low densitylipoprotein receptor LDLRAD2 ENSG00000187942 Low density lipoproteinreceptor class A domain containing 2 LEAP2 ENSG00000164406 Liverexpressed antimicrobial peptide 2 LECT2 ENSG00000145826 Leukocytecell-derived chemotaxin 2 LEFTY1 ENSG00000243709 Left-rightdetermination factor 1 LEFTY2 ENSG00000143768 Left-right determinationfactor 2 LEP ENSG00000174697 Leptin LFNG ENSG00000106003 LFNGO-fucosylpeptide 3-beta-N-acetylglucosaminyltransferase LGALS3BPENSG00000108679 Lectin, galactoside-binding, soluble, 3 binding proteinLGI1 ENSG00000108231 Leucine-rich, glioma inactivated 1 LGI2ENSG00000153012 Leucine-rich repeat LGI family, member 2 LGI3ENSG00000168481 Leucine-rich repeat LGI family, member 3 LGI4ENSG00000153902 Leucine-rich repeat LGI family, member 4 LGMNENSG00000100600 Legumain LGR4 ENSG00000205213 Leucine-rich repeatcontaining G protein-coupled receptor 4 LHB ENSG00000104826 Luteinizinghormone beta polypeptide LHCGR ENSG00000138039 Luteinizinghormone/choriogonadotropin receptor LIF ENSG00000128342 Leukemiainhibitory factor LIFR ENSG00000113594 Leukemia inhibitory factorreceptor alpha LILRA1 ENSG00000104974 Leukocyte immunoglobulin-likereceptor, subfamily A (with TM domain), member 1 LILRA2 ENSG00000239998Leukocyte immunoglobulin-like receptor, subfamily A (with TM domain),member 2 LILRB3 ENSG00000204577 Leukocyte immunoglobulin-like receptor,subfamily B (with TM and ITIM domains), member 3 LIME1 ENSG00000203896Lck interacting transmembrane adaptor 1 LING01 ENSG00000169783 Leucinerich repeat and Ig domain containing 1 LIPA ENSG00000107798 Lipase A,lysosomal acid, cholesterol esterase LIPC ENSG00000166035 Lipase,hepatic LIPF ENSG00000182333 Lipase, gastric LIPG ENSG00000101670Lipase, endothelial LIPH ENSG00000163898 Lipase, member H LIPKENSG00000204021 Lipase, family member K LIPM ENSG00000173239 Lipase,family member M LIPN ENSG00000204020 Lipase, family member N LMAN2ENSG00000169223 Lectin, mannose-binding 2 LMNTD1 ENSG00000152936 Lamintail domain containing 1 LNX1 ENSG00000072201 Ligand of numb-protein X1, E3 ubiquitin protein ligase LOX ENSG00000113083 Lysyl oxidase LOXL1ENSG00000129038 Lysyl oxidase-like 1 LOXL2 ENSG00000134013 Lysyloxidase-like 2 LOXL3 ENSG00000115318 Lysyl oxidase-like 3 LOXL4ENSG00000138131 Lysyl oxidase-like 4 LPA ENSG00000198670 Lipoprotein,Lp(a) LPL ENSG00000175445 Lipoprotein lipase LPO ENSG00000167419Lactoperoxidase LRAT ENSG00000121207 Lecithin retinol acyltransferase(phosphatidylcholine--retinol O- acyltransferase) LRCH3 ENSG00000186001Leucine-rich repeats and calponin homology (CH) domain containing 3LRCOL1 ENSG00000204583 Leucine rich colipase-like 1 LRFN4ENSG00000173621 Leucine rich repeat and fibronectin type III domaincontaining 4 LRFN5 ENSG00000165379 Leucine rich repeat and fibronectintype III domain containing 5 LRG1 ENSG00000171236 Leucine-richalpha-2-glycoprotein 1 LRP1 ENSG00000123384 Low density lipoproteinreceptor-related protein 1 LRP11 ENSG00000120256 Low density lipoproteinreceptor-related protein 11 LRP1B ENSG00000168702 Low densitylipoprotein receptor-related protein 1B LRP2 ENSG00000081479 Low densitylipoprotein receptor-related protein 2 LRP4 ENSG00000134569 Low densitylipoprotein receptor-related protein 4 LRPAP1 ENSG00000163956 Lowdensity lipoprotein receptor-related protein associated protein 1 LRRC17ENSG00000128606 Leucine rich repeat containing 17 LRRC32 ENSG00000137507Leucine rich repeat containing 32 LRRC3B ENSG00000179796 Leucine richrepeat containing 3B LRRC4B ENSG00000131409 Leucine rich repeatcontaining 4B LRRC70 ENSG00000186105 Leucine rich repeat containing 70LRRN3 ENSG00000173114 Leucine rich repeat neuronal 3 LRRTM1ENSG00000162951 Leucine rich repeat transmembrane neuronal 1 LRRTM2ENSG00000146006 Leucine rich repeat transmembrane neuronal 2 LRRTM4ENSG00000176204 Leucine rich repeat transmembrane neuronal 4 LRTM2ENSG00000166159 Leucine-rich repeats and transmembrane domains 2 LSRENSG00000105699 Lipolysis stimulated lipoprotein receptor LST1ENSG00000204482 Leukocyte specific transcript 1 LTA ENSG00000226979Lymphotoxin alpha LTBP1 ENSG00000049323 Latent transforming growthfactor beta binding protein 1 LTBP2 ENSG00000119681 Latent transforminggrowth factor beta binding protein 2 LTBP3 ENSG00000168056 Latenttransforming growth factor beta binding protein 3 LTBP4 ENSG00000090006Latent transforming growth factor beta binding protein 4 LTBRENSG00000111321 Lymphotoxin beta receptor (TNFR superfamily, member 3)LTF ENSG00000012223 Lactotransferrin LTK ENSG00000062524 Leukocytereceptor tyrosine kinase LUM ENSG00000139329 Lumican LUZP2ENSG00000187398 Leucine zipper protein 2 LVRN ENSG00000172901 LaeverinLY6E ENSG00000160932 Lymphocyte antigen 6 complex, locus E LY6G5BENSG00000240053 Lymphocyte antigen 6 complex, locus G5B LY6G6DENSG00000244355 Lymphocyte antigen 6 complex, locus G6D LY6G6EENSG00000255552 Lymphocyte antigen 6 complex, locus G6E (pseudogene)LY6H ENSG00000176956 Lymphocyte antigen 6 complex, locus H LY6KENSG00000160886 Lymphocyte antigen 6 complex, locus K LY86ENSG00000112799 Lymphocyte antigen 86 LY96 ENSG00000154589 Lymphocyteantigen 96 LYG1 ENSG00000144214 Lysozyme G-like 1 LYG2 ENSG00000185674Lysozyme G-like 2 LYNX1 ENSG00000180155 Ly6/neurotoxin 1 LYPD1ENSG00000150551 LY6/PLAUR domain containing 1 LYPD2 ENSG00000197353LY6/PLAUR domain containing 2 LYPD4 ENSG00000273111 LY6/PLAUR domaincontaining 4 LYPD6 ENSG00000187123 LY6/PLAUR domain containing 6 LYPD6BENSG00000150556 LY6/PLAUR domain containing 6B LYPD8 ENSG00000259823LY6/PLAUR domain containing 8 LYZ ENSG00000090382 Lysozyme LYZL4ENSG00000157093 Lysozyme-like 4 LYZL6 ENSG00000275722 Lysozyme-like 6M6PR ENSG00000003056 Mannose-6-phosphate receptor (cation dependent)MAD1L1 ENSG00000002822 MAD1 mitotic arrest deficient-like 1 (yeast) MAGENSG00000105695 Myelin associated glycoprotein MAGT1 ENSG00000102158Magnesium transporter 1 MALSU1 ENSG00000156928 Mitochondrial assembly ofribosomal large subunit 1 MAMDC2 ENSG00000165072 MAM domain containing 2MAN2B1 ENSG00000104774 Mannosidase, alpha, class 2B, member 1 MAN2B2ENSG00000013288 Mannosidase, alpha, class 2B, member 2 MANBAENSG00000109323 Mannosidase, beta A, lysosomal MANEAL ENSG00000185090Mannosidase, endo-alpha-like MANF ENSG00000145050 Mesencephalicastrocyte-derived neurotrophic factor MANSC1 ENSG00000111261 MANSCdomain containing 1 MAP3K9 ENSG00000006432 Mitogen-activated proteinkinase 9 MASP1 ENSG00000127241 Mannan-binding lectin serine peptidase 1(C4/C2 activating component of Ra-reactive factor) MASP2 ENSG00000009724Mannan-binding lectin serine peptidase 2 MATN1 ENSG00000162510 Matrilin1, cartilage matrix protein MATN2 ENSG00000132561 Matrilin 2 MATN3ENSG00000132031 Matrilin 3 MATN4 ENSG00000124159 Matrilin 4 MATR3ENSG00000015479 Matrin 3 MATR3 ENSG00000280987 Matrin 3 MAU2ENSG00000129933 MAU2 sister chromatid cohesion factor MAZENSG00000103495 MYC-associated zinc finger protein (purine-bindingtranscription factor) MBD6 ENSG00000166987 Methyl-CpG binding domainprotein 6 MBL2 ENSG00000165471 Mannose-binding lectin (protein C) 2,soluble MBNL1 ENSG00000152601 Muscleblind-like splicing regulator 1MCCC1 ENSG00000078070 Methylcrotonoyl-CoA carboxylase 1 (alpha) MCCD1ENSG00000204511 Mitochondrial coiled-coil domain 1 MCEE ENSG00000124370Methylmalonyl CoA epimerase MCF2L ENSG00000126217 MCF.2 cell linederived transforming sequence-like MCFD2 ENSG00000180398 Multiplecoagulation factor deficiency 2 MDFIC ENSG00000135272 MyoD familyinhibitor domain containing MDGA1 ENSG00000112139 MAM domain containingglycosylphosphatidylinositol anchor 1 MDK ENSG00000110492 Midkine(neurite growth-promoting factor 2) MED20 ENSG00000124641 Mediatorcomplex subunit 20 MEGF10 ENSG00000145794 Multiple EGF-like-domains 10MEGF6 ENSG00000162591 Multiple EGF-like-domains 6 MEI1 ENSG00000167077Meiotic double-stranded break formation protein 1 MEI4 ENSG00000269964Meiotic double-stranded break formation protein 4 MEIS1 ENSG00000143995Meis homeobox 1 MEIS3 ENSG00000105419 Meis homeobox 3 MEPEENSG00000152595 Matrix extracellular phosphoglycoprotein MESDC2ENSG00000117899 Mesoderm development candidate 2 WIEST ENSG00000106484Mesoderm specific transcript MET ENSG00000105976 MET proto-oncogene,receptor tyrosine kinase METRN ENSG00000103260 Meteorin, glial celldifferentiation regulator METRNL ENSG00000176845 Meteorin, glial celldifferentiation regulator-like METTL17 ENSG00000165792 Methyltransferaselike 17 METTL24 ENSG00000053328 Methyltransferase like 24 METTL7BENSG00000170439 Methyltransferase like 7B METTL9 ENSG00000197006Methyltransferase like 9 MEX3C ENSG00000176624 Mex-3 RNA binding familymember C MFAP2 ENSG00000117122 Microfibrillar-associated protein 2 MFAP3ENSG00000037749 Microfibrillar-associated protein 3 MFAP3LENSG00000198948 Microfibrillar-associated protein 3-like MFAP4ENSG00000166482 Microfibrillar-associated protein 4 MFAP5ENSG00000197614 Microfibrillar associated protein 5 MFGE8ENSG00000140545 Milk fat globule-EGF factor 8 protein MFI2ENSG00000163975 Antigen p97 (melanoma associated) identified bymonoclonal antibodies 133.2 and 96.5 MFNG ENSG00000100060 MFNGO-fucosylpeptide 3-beta-N-acetylglucosaminyltransferase MGAENSG00000174197 MGA, MAX dimerization protein MGAT2 ENSG00000168282Mannosyl (alpha-1,6-)-glycoprotein beta-1,2-N-acetylglucosaminyltransferase MGAT3 ENSG00000128268 Mannosyl(beta-1,4-)-glycoprotein beta-1, 4-N-acetylglucosaminyltransferaseMGAT4A ENSG00000071073 Mannosyl (alpha-1,3-)-glycoprotein beta-1,4-N-acetylglucosaminyltransferase, isozyme A MGAT4B ENSG00000161013Mannosyl (alpha-1,3-)-glycoprotein beta-1,4-N-acetylglucosaminyltransferase, isozyme B MGAT4D ENSG00000205301MGAT4 family, member D MGLL ENSG00000074416 Monoglyceride lipase MGPENSG00000111341 Matrix Gla protein MGST2 ENSG00000085871 Microsomalglutathione S-transferase 2 MIA ENSG00000261857 Melanoma inhibitoryactivity MIA2 ENSG00000150526 Melanoma inhibitory activity 2 MIA3ENSG00000154305 Melanoma inhibitory activity family, member 3 MICUlENSG00000107745 Mitochondrial calcium uptake 1 MIER1 ENSG00000198160Mesoderm induction early response 1, transcriptional regulatorMINOS1-NBL1 ENSG00000270136 MINOS1-NBL1 readthrough MINPP1ENSG00000107789 Multiple inositol-polyphosphate phosphatase 1 MLECENSG00000110917 Malectin MLN ENSG00000096395 Motilin MLXIPENSG00000175727 MLX interacting protein MLXIPL ENSG00000009950 MLXinteracting protein-like MMP1 ENSG00000196611 Matrix metallopeptidase 1MMP10 ENSG00000166670 Matrix metallopeptidase 10 MMP11 ENSG00000099953Matrix metallopeptidase 11 MMP12 ENSG00000262406 Matrix metallopeptidase12 MMP13 ENSG00000137745 Matrix metallopeptidase 13 MMP14ENSG00000157227 Matrix metallopeptidase 14 (membrane-inserted) MMP17ENSG00000198598 Matrix metallopeptidase 17 (membrane-inserted) MMP19ENSG00000123342 Matrix metallopeptidase 19 MMP2 ENSG00000087245 Matrixmetallopeptidase 2 MMP20 ENSG00000137674 Matrix metallopeptidase 20MMP21 ENSG00000154485 Matrix metallopeptidase 21 MMP25 ENSG00000008516Matrix metallopeptidase 25 MMP26 ENSG00000167346 Matrix metallopeptidase26 MMP27 ENSG00000137675 Matrix metallopeptidase 27 MMP28ENSG00000271447 Matrix metallopeptidase 28 MMP3 ENSG00000149968 Matrixmetallopeptidase 3 MMP7 ENSG00000137673 Matrix metallopeptidase 7 MMP8ENSG00000118113 Matrix metallopeptidase 8 MMP9 ENSG00000100985 Matrixmetallopeptidase 9 MMRN1 ENSG00000138722 Multimerin 1 MMRN2ENSG00000173269 Multimerin 2 MOXD1 ENSG00000079931 Monooxygenase,DBH-like 1 MPO ENSG00000005381 Myeloperoxidase MPPED1 ENSG00000186732Metallophosphoesterase domain containing 1 MPZL1 ENSG00000197965 Myelinprotein zero-like 1 MR1 ENSG00000153029 Major histocompatibilitycomplex, class I-related MRPL2 ENSG00000112651 Mitochondrial ribosomalprotein L2 MRPL21 ENSG00000197345 Mitochondrial ribosomal protein L21MRPL22 ENSG00000082515 Mitochondrial ribosomal protein L22 MRPL24ENSG00000143314 Mitochondrial ribosomal protein L24 MRPL27ENSG00000108826 Mitochondrial ribosomal protein L27 MRPL32ENSG00000106591 Mitochondrial ribosomal protein L32 MRPL34ENSG00000130312 Mitochondrial ribosomal protein L34 MRPL35ENSG00000132313 Mitochondrial ribosomal protein L35 MRPL52ENSG00000172590 Mitochondrial ribosomal protein L52 MRPL55ENSG00000162910 Mitochondrial ribosomal protein L55 MRPS14ENSG00000120333 Mitochondrial ribosomal protein S14 MRPS22ENSG00000175110 Mitochondrial ribosomal protein S22 MRPS28ENSG00000147586 Mitochondrial ribosomal protein S28 MS4A14ENSG00000166928 Membrane-spanning 4-domains, subfamily A, member 14MS4A3 ENSG00000149516 Membrane-spanning 4-domains, subfamily A, member 3(hematopoietic cell-specific) MSH3 ENSG00000113318 MutS homolog 3 MSH5ENSG00000204410 MutS homolog 5 MSLN ENSG00000102854 Mesothelin MSMBENSG00000263639 Microseminoprotein, beta- MSRA ENSG00000175806Methionine sulfoxide reductase A MSRB2 ENSG00000148450 Methioninesulfoxide reductase B2 MSRB3 ENSG00000174099 Methionine sulfoxidereductase B3 MST1 ENSG00000173531 Macrophage stimulating 1 MSTNENSG00000138379 Myostatin MT1G ENSG00000125144 Metallothionein 1G MTHFD2ENSG00000065911 Methylenetetrahydrofolate dehydrogenase (NADP +dependent) 2, methenyltetrahydrofolate cyclohydrolase MTMR14ENSG00000163719 Myotubularin related protein 14 MTRNR2L11ENSG00000270188 MT-RNR2-like 11 (pseudogene) MTRR ENSG000001242755-methyltetrahydrofolate-homocysteine methyltransferase reductase MTTPENSG00000138823 Microsomal triglyceride transfer protein MTX2ENSG00000128654 Metaxin 2 MUC1 ENSG00000185499 Mucin 1, cell surfaceassociated MUC13 ENSG00000173702 Mucin 13, cell surface associated MUC20ENSG00000176945 Mucin 20, cell surface associated MUC3A ENSG00000169894Mucin 3A, cell surface associated MUC5AC ENSG00000215182 Mucin 5AC,oligomeric mucus/gel-forming MUC5B ENSG00000117983 Mucin 5B, oligomericmucus/gel-forming MUC6 ENSG00000184956 Mucin 6, oligomericmucus/gel-forming MUC7 ENSG00000171195 Mucin 7, secreted MUCL1ENSG00000172551 Mucin-like 1 MXRA5 ENSG00000101825 Matrix-remodellingassociated 5 MXRA7 ENSG00000182534 Matrix-remodelling associated 7 MYDGFENSG00000074842 Myeloid-derived growth factor MYL1 ENSG00000168530Myosin, light chain 1, alkali; skeletal, fast MYOC ENSG00000034971Myocilin, trabecular meshwork inducible glucocorticoid response MYRFLENSG00000166268 Myelin regulatory factor-like MZB1 ENSG00000170476Marginal zone B and B1 cell-specific protein N4BP2L2 ENSG00000244754NEDD4 binding protein 2-like 2 NAA38 ENSG00000183011N(alpha)-acetyltransferase 38, NatC auxiliary subunit NAAAENSG00000138744 N-acylethanolamine acid amidase NAGA ENSG00000198951N-acetylgalactosaminidase, alpha- NAGLU ENSG00000108784N-acetylglucosaminidase, alpha NAGS ENSG00000161653 N-acetylglutamatesynthase NAPSA ENSG00000131400 Napsin A aspartic peptidase NBL1ENSG00000158747 Neuroblastoma 1, DAN family BMP antagonist NCAM1ENSG00000149294 Neural cell adhesion molecule 1 NCAN ENSG00000130287Neurocan NCBP2-A52 ENSG00000270170 NCBP2 antisense RNA 2 (head to head)NCSTN ENSG00000162736 Nicastrin NDNF ENSG00000173376 Neuron-derivedneurotrophic factor NDP ENSG00000124479 Norrie disease (pseudoglioma)NDUFA10 ENSG00000130414 NADH dehydrogenase (ubiquinone) 1 alphasubcomplex, 10, 42 kDa NDUFB5 ENSG00000136521 NADH dehydrogenase(ubiquinone) 1 beta subcomplex, 5, 16 kDa NDUFS8 ENSG00000110717 NADHdehydrogenase (ubiquinone) Fe-S protein 8, 23 kDa (NADH-coenzyme Qreductase) NDUFV1 ENSG00000167792 NADH dehydrogenase (ubiquinone)flavoprotein 1, 51 kDa NECAB3 ENSG00000125967 N-terminal EF-hand calciumbinding protein 3 NELL1 ENSG00000165973 Neural EGFL like 1 NELL2ENSG00000184613 Neural EGFL like 2 NENF ENSG00000117691 Neudesinneurotrophic factor NETO1 ENSG00000166342 Neuropilin (NRP) and tolloid(TLL)-like 1 NFASC ENSG00000163531 Neurofascin NFE2L1 ENSG00000082641Nuclear factor, erythroid 2-like 1 NFE2L3 ENSG00000050344 Nuclearfactor, erythroid 2-like 3 NGEF ENSG00000066248 Neuronal guaninenucleotide exchange factor NGF ENSG00000134259 Nerve growth factor (betapolypeptide) NGLY1 ENSG00000151092 N-glycanase 1 NGRN ENSG00000182768Neugrin, neurite outgrowth associated NHLRC3 ENSG00000188811 NHL repeatcontaining 3 NID 1 ENSG00000116962 Nidogen 1 NID2 ENSG00000087303Nidogen 2 (osteonidogen) NKG7 ENSG00000105374 Natural killer cellgranule protein 7 NLGN3 ENSG00000196338 Neuroligin 3 NLGN4YENSG00000165246 Neuroligin 4, Y-linked NLRP5 ENSG00000171487 NLR family,pyrin domain containing 5 NMB ENSG00000197696 Neuromedin B NME1ENSG00000239672 NME/NM23 nucleoside diphosphate kinase 1 NME1-NME2ENSG00000011052 NME1-NME2 readthrough NME3 ENSG00000103024 NME/NM23nucleoside diphosphate kinase 3 NMS ENSG00000204640 Neuromedin S NMUENSG00000109255 Neuromedin U NOA1 ENSG00000084092 Nitric oxideassociated 1 NODAL ENSG00000156574 Nodal growth differentiation factorNOG ENSG00000183691 Noggin NOM03 ENSG00000103226 NODAL modulator 3NOS1AP ENSG00000198929 Nitric oxide synthase 1 (neuronal) adaptorprotein NOTCH3 ENSG00000074181 Notch 3 NOTUM ENSG00000185269 Notumpectinacetylesterase homolog (Drosophila) NOV ENSG00000136999Nephroblastoma overexpressed NPB ENSG00000183979 Neuropeptide B NPC2ENSG00000119655 Niemann-Pick disease, type C2 NPFF ENSG00000139574Neuropeptide FF-amide peptide precursor NPFFR2 ENSG00000056291Neuropeptide FF receptor 2 NPH S1 ENSG00000161270 Nephrosis 1,congenital, Finnish type (nephrin) NPNT ENSG00000168743 NephronectinNPPA ENSG00000175206 Natriuretic peptide A NPPB ENSG00000120937Natriuretic peptide B NPPC ENSG00000163273 Natriuretic peptide C NPSENSG00000214285 Neuropeptide S NPTX1 ENSG00000171246 Neuronal pentraxinI NPTX2 ENSG00000106236 Neuronal pentraxin II NPTXR ENSG00000221890Neuronal pentraxin receptor NPVF ENSG00000105954 Neuropeptide VFprecursor NPW ENSG00000183971 Neuropeptide W NPY ENSG00000122585Neuropeptide Y NQ02 ENSG00000124588 NAD(P)H dehydrogenase, quinone 2NRCAM ENSG00000091129 Neuronal cell adhesion molecule NRG1ENSG00000157168 Neuregulin 1 NRN1L ENSG00000188038 Neuritin 1-like NRP1ENSG00000099250 Neuropilin 1 NRP2 ENSG00000118257 Neuropilin 2 NRTNENSG00000171119 Neurturin NRXN1 ENSG00000179915 Neurexin 1 NRXN2ENSG00000110076 Neurexin 2 NT5C3A ENSG00000122643 5′-nucleotidase,cytosolic IIIA NT5DC3 ENSG00000111696 5′-nucleotidase domain containing3 NT5E ENSG00000135318 5′-nucleotidase, ecto (CD73) NTF3 ENSG00000185652Neurotrophin 3 NTF4 ENSG00000225950 Neurotrophin 4 NTM ENSG00000182667Neurotrimin NTN1 ENSG00000065320 Netrin 1 NTN3 ENSG00000162068 Netrin 3NTN4 ENSG00000074527 Netrin 4 NTN5 ENSG00000142233 Netrin 5 NTNG1ENSG00000162631 Netrin G1 NTNG2 ENSG00000196358 Netrin G2 NTSENSG00000133636 Neurotensin NUBPL ENSG00000151413 Nucleotide bindingprotein-like NUCB1 ENSG00000104805 Nucleobindin 1 NUCB2 ENSG00000070081Nucleobindin 2 NUDT19 ENSG00000213965 Nudix (nucleoside diphosphatelinked moiety X)-type motif 19 NUDT9 ENSG00000170502 Nudix (nucleosidediphosphate linked moiety X)-type motif 9 NUP155 ENSG00000113569Nucleoporin 155 kDa NUP214 ENSG00000126883 Nucleoporin 214 kDa NUP85ENSG00000125450 Nucleoporin 85 kDa NXPE3 ENSG00000144815 Neurexophilinand PC-esterase domain family, member 3 NXPE4 ENSG00000137634Neurexophilin and PC-esterase domain family, member 4 NXPH1ENSG00000122584 Neurexophilin 1 NXPH2 ENSG00000144227 Neurexophilin 2NXPH3 ENSG00000182575 Neurexophilin 3 NXPH4 ENSG00000182379Neurexophilin 4 NYX ENSG00000188937 Nyctalopin OAF ENSG00000184232 Outat first homolog OBP2A ENSG00000122136 Odomnt binding protein 2A OBP2BENSG00000171102 Odomnt binding protein 2B OC90 ENSG00000253117 Otoconin90 OCLN ENSG00000197822 Occludin ODAM ENSG00000109205 Odontogenic,ameloblast asssociated OGG1 ENSG00000114026 8-oxoguanine DNA glycosylaseOGN ENSG00000106809 Osteoglycin O1T3 ENSG00000138315 Oncoprotein inducedtranscript 3 OLFM1 ENSG00000130558 Olfactomedin 1 OLFM2 ENSG00000105088Olfactomedin 2 OLFM3 ENSG00000118733 Olfactomedin 3 OLFM4ENSG00000102837 Olfactomedin 4 OLFML1 ENSG00000183801 Olfactomedin-like1 OLFML2A ENSG00000185585 Olfactomedin-like 2A OLFML2B ENSG00000162745Olfactomedin-like 2B OLFML3 ENSG00000116774 Olfactomedin-like 3 OMDENSG00000127083 Osteomodulin OMG ENSG00000126861 Oligodendrocyte myelinglycoprotein OOSP2 ENSG00000149507 Oocyte secreted protein 2 OPCMLENSG00000183715 Opioid binding protein/cell adhesion molecule-like OPTCENSG00000188770 Opticin ORAI1 ENSG00000276045 ORAI calciumrelease-activated calcium modulator 1 ORM1 ENSG00000229314 Orosomucoid 1ORM2 ENSG00000228278 Orosomucoid 2 ORMDL2 ENSG00000123353 ORMDLsphingolipid biosynthesis regulator 2 OS9 ENSG00000135506 Osteosarcomaamplified 9, endoplasmic reticulum lectin OSCAR ENSG00000170909Osteoclast associated, immunoglobulin-like receptor OSM ENSG00000099985Oncostatin M OSMR ENSG00000145623 Oncostatin M receptor OSTNENSG00000188729 Osteocrin OTOA ENSG00000155719 Otoancorin OTOGENSG00000188162 Otogelin OTOGL ENSG00000165899 Otogelin-like OTOL1ENSG00000182447 Otolin 1 OTOR ENSG00000125879 Otoraplin OTOSENSG00000178602 Otospiralin OVCH1 ENSG00000187950 Ovochymase 1 OVCH2ENSG00000183378 Ovochymase 2 (gene/pseudogene) OVGP1 ENSG00000085465Oviductal glycoprotein 1, 120 kDa OXCT1 ENSG00000083720 3-oxoacid CoAtransferase 1 OXCT2 ENSG00000198754 3-oxoacid CoA transferase 2 OXNAD1ENSG00000154814 Oxidoreductase NAD-binding domain containing 1 OXTENSG00000101405 Oxytocin/neurophysin I prepropeptide P3H1ENSG00000117385 Prolyl 3-hydroxylase 1 P3H2 ENSG00000090530 Prolyl3-hydroxylase 2 P3H3 ENSG00000110811 Prolyl 3-hydroxylase 3 P3H4ENSG00000141696 Prolyl 3-hydroxylase family member 4 (non-enzymatic)P4HA1 ENSG00000122884 Prolyl 4-hydroxylase, alpha polypeptide I P4HA2ENSG00000072682 Prolyl 4-hydroxylase, alpha polypeptide II P4HA3ENSG00000149380 Prolyl 4-hydroxylase, alpha polypeptide III P4HBENSG00000185624 Prolyl 4-hydroxylase, beta polypeptide PAEPENSG00000122133 Progestagen-associated endometrial protein PAMENSG00000145730 Peptidylglycine alpha-amidating monooxygenase PAMR1ENSG00000149090 Peptidase domain containing associated with muscleregeneration 1 PAPL ENSG00000183760 Iron/zinc purple acidphosphatase-like protein PAPLN ENSG00000100767 Papilin,proteoglycan-like sulfated glycoprotein PAPPA ENSG00000182752Pregnancy-associated plasma protein A, pappalysin 1 PAPPA2ENSG00000116183 Pappalysin 2 PARP15 ENSG00000173200 Poly (ADP-ribose)polymerase family, member 15 PARVB ENSG00000188677 Parvin, beta PA1E1ENSG00000171053 Prostate and testis expressed 1 PA1E2 ENSG00000196844Prostate and testis expressed 2 PA1E3 ENSG00000236027 Prostate andtestis expressed 3 PA1E4 ENSG00000237353 Prostate and testis expressed 4PATL2 ENSG00000229474 Protein associated with topoisomerase II homolog 2(yeast) PAX2 ENSG00000075891 Paired box 2 PAX4 ENSG00000106331 Pairedbox 4 PCCB ENSG00000114054 Propionyl CoA calboxylase, beta polypeptidePCDH1 ENSG00000156453 Protocadherin 1 PCDH12 ENSG00000113555Protocadherin 12 PCDH15 ENSG00000150275 Protocadherin-related 15 PCDHA1ENSG00000204970 Protocadherin alpha 1 PCDHA10 ENSG00000250120Protocadherin alpha 10 PCDHA11 ENSG00000249158 Protocadherin alpha 11PCDHA6 ENSG00000081842 Protocadherin alpha 6 PCDHB12 ENSG00000120328Protocadherin beta 12 PCDHGA11 ENSG00000253873 Protocadherin gammasubfamily A, 11 PCF11 ENSG00000165494 PCF11 cleavage and polyadenylationfactor subunit PCOLCE ENSG00000106333 Procollagen C-endopeptidaseenhancer PCOLCE2 ENSG00000163710 Procollagen C-endopeptidase enhancer 2PCSK1 ENSG00000175426 Proprotein convertase subtilisin/kexin type 1PCSK1N ENSG00000102109 Proprotein convertase subtilisin/kexin type 1inhibitor PCSK2 ENSG00000125851 Proprotein convertase subtilisin/kexintype 2 PCSK4 ENSG00000115257 Proprotein convertase subtilisin/kexin type4 PCSK5 ENSG00000099139 Proprotein convertase subtilisin/kexin type 5PCSK9 ENSG00000169174 Proprotein convertase subtilisin/kexin type 9PCYOX1 ENSG00000116005 Prenylcysteine oxidase 1 PCYOX1L ENSG00000145882Prenylcysteine oxidase 1 like PDDC1 ENSG00000177225 Parkinson disease 7domain containing 1 PDE11A ENSG00000128655 Phosphodiesterase 11A PDE2AENSG00000186642 Phosphodiesterase 2A, cGMP-stimulated PDE7AENSG00000205268 Phosphodiesterase 7A PDF ENSG00000258429 Peptidedeformylase (mitochondrial) PDGFA ENSG00000197461 Platelet-derivedgrowth factor alpha polypeptide PDGFB ENSG00000100311 Platelet-derivedgrowth factor beta polypeptide PDGFC ENSG00000145431 Platelet derivedgrowth factor C PDGFD ENSG00000170962 Platelet derived growth factor DPDGFRA ENSG00000134853 Platelet-derived growth factor receptor, alphapolypeptide PDGFRB ENSG00000113721 Platelet-derived growth factorreceptor, beta polypeptide PDGFRL ENSG00000104213 Platelet-derivedgrowth factor receptor-like PDHAl ENSG00000131828 Pyruvate dehydrogenase(lipoamide) alpha 1 PDIA2 ENSG00000185615 Protein disulfide isomerasefamily A, member 2 PDIA3 ENSG00000167004 Protein disulfide isomerasefamily A, member 3 PDIA4 ENSG00000155660 Protein disulfide isomerasefamily A, member 4 PDIA5 ENSG00000065485 Protein disulfide isomerasefamily A, member 5 PDIA6 ENSG00000143870 Protein disulfide isomerasefamily A, member 6 PDILT ENSG00000169340 Protein disulfideisomerase-like, testis expressed PDYN ENSG00000101327 Prodynorphin PDZD8ENSG00000165650 PDZ domain containing 8 PDZRN4 ENSG00000165966 PDZdomain containing ring finger 4 PEAR1 ENSG00000187800 Plateletendothelial aggregation receptor 1 PEBP4 ENSG00000134020Phosphatidylethanolamine-binding protein 4 PECAM1 ENSG00000261371Platelet/endothelial cell adhesion molecule 1 PENK ENSG00000181195Proenkephalin PET117 ENSG00000232838 PET117 homolog PF4 ENSG00000163737Platelet factor 4 PF4V1 ENSG00000109272 Platelet factor 4 variant 1 PFKPENSG00000067057 Phosphofructokinase, platelet PFN1 ENSG00000108518Profilin 1 PGA3 ENSG00000229859 Pepsinogen 3, group I (pepsinogen A)PGA4 ENSG00000229183 Pepsinogen 4, group I (pepsinogen A) PGA5ENSG00000256713 Pepsinogen 5, group I (pepsinogen A) PGAM5ENSG00000247077 PGAM family member 5, serine/threonine proteinphosphatase, mitochondrial PGAP3 ENSG00000161395 Post-GPI attachment toproteins 3 PGC ENSG00000096088 Progastricsin (pepsinogen C) PGFENSG00000119630 Placental growth factor PGLYRP1 ENSG00000008438Peptidoglycan recognition protein 1 PGLYRP2 ENSG00000161031Peptidoglycan recognition protein 2 PGLYRP3 ENSG00000159527Peptidoglycan recognition protein 3 PGLYRP4 ENSG00000163218Peptidoglycan recognition protein 4 PHACTR1 ENSG00000112137 Phosphataseand actin regulator 1 PHB ENSG00000167085 Prohibitin PI15ENSG00000137558 Peptidase inhibitor 15 PI3 ENSG00000124102 Peptidaseinhibitor 3, skin-derived PIANP ENSG00000139200 PILR alpha associatedneural protein PIGK ENSG00000142892 Phosphatidylinositol glycan anchorbiosynthesis, class K PIGL ENSG00000108474 Phosphatidylinositol glycananchor biosynthesis, class L PIGT ENSG00000124155 Phosphatidylinositolglycan anchor biosynthesis, class T PIGZ ENSG00000119227Phosphatidylinositol glycan anchor biosynthesis, class Z PIK3AP1ENSG00000155629 Phosphoinositide-3-kinase adaptor protein 1 PIK3IP1ENSG00000100100 Phosphoinositide-3-kinase interacting protein 1 PILRAENSG00000085514 Paired immunoglobin-like type 2 receptor alpha PILRBENSG00000121716 Paired immunoglobin-like type 2 receptor beta PINLYPENSG00000234465 Phospholipase A2 inhibitor and LY6/PLAUR domaincontaining PIP ENSG00000159763 Prolactin-induced protein PIWIL4ENSG00000134627 Piwi-like RNA-mediated gene silencing 4 PKDCCENSG00000162878 Protein kinase domain containing, cytoplasmic PKHD1ENSG00000170927 Polycystic kidney and hepatic disease 1 (autosomalrecessive) PLA1A ENSG00000144837 Phospholipase A1 member A PLA2G10ENSG00000069764 Phospholipase A2, group X PLA2G12A ENSG00000123739Phospholipase A2, group XIIA PLA2G12B ENSG00000138308 Phospholipase A2,group XIIB PLA2G15 ENSG00000103066 Phospholipase A2, group XV PLA2G1BENSG00000170890 Phospholipase A2, group IB (pancreas) PLA2G2AENSG00000188257 Phospholipase A2, group IIA (platelets, synovial fluid)PLA2G2C ENSG00000187980 Phospholipase A2, group IIC PLA2G2DENSG00000117215 Phospholipase A2, group IID PLA2G2E ENSG00000188784Phospholipase A2, group IIE PLA2G3 ENSG00000100078 Phospholipase A2,group III PLA2G5 ENSG00000127472 Phospholipase A2, group V PLA2G7ENSG00000146070 Phospholipase A2, group VII (platelet-activating factoracetylhydrolase, plasma) PLA2R1 ENSG00000153246 Phospholipase A2receptor 1, 180 kDa PLAC1 ENSG00000170965 Placenta-specific 1 PLAC9ENSG00000189129 Placenta-specific 9 PLAT ENSG00000104368 Plasminogenactivator, tissue PLAU ENSG00000122861 Plasminogen activator, urokinasePLAUR ENSG00000011422 Plasminogen activator, urokinase receptor PLBD1ENSG00000121316 Phospholipase B domain containing 1 PLBD2ENSG00000151176 Phospholipase B domain containing 2 PLG ENSG00000122194Plasminogen PLGLB1 ENSG00000183281 Plasminogen-like B1 PLGLB2ENSG00000125551 Plasminogen-like B2 PLOD1 ENSG00000083444Procollagen-lysine, 2-oxoglutarate 5-dioxygenase 1 PLOD2 ENSG00000152952Procollagen-lysine, 2-oxoglutarate 5-dioxygenase 2 PLOD3 ENSG00000106397Procollagen-lysine, 2-oxoglutarate 5-dioxygenase 3 PLTP ENSG00000100979Phospholipid transfer protein PLXNA4 ENSG00000221866 Plexin A4 PLXNB2ENSG00000196576 Plexin B2 PM20D1 ENSG00000162877 Peptidase M20 domaincontaining 1 PMCH ENSG00000183395 Pro-melanin-concentrating hormone PMELENSG00000185664 Premelanosome protein PMEPA1 ENSG00000124225 Prostatetransmembrane protein, androgen induced 1 PNLIP ENSG00000175535Pancreatic lipase PNLIPRP1 ENSG00000187021 Pancreatic lipase-relatedprotein 1 PNLIPRP3 ENSG00000203837 Pancreatic lipase-related protein 3PNOC ENSG00000168081 Prepronociceptin PNP ENSG00000198805 Purinenucleoside phosphorylase PNPLA4 ENSG00000006757 Patatin-likephospholipase domain containing 4 PODNL1 ENSG00000132000 Podocan-like 1POFUT1 ENSG00000101346 Protein O-fucosyltransferase 1 POFUT2ENSG00000186866 Protein O-fucosyltransferase 2 POGLUT1 ENSG00000163389Protein O-glucosyltransferase 1 POLL ENSG00000166169 Polymerase (DNAdirected), lambda POMC ENSG00000115138 Proopiomelanocortin POMGNT2ENSG00000144647 Protein O-linked mannose N-acetylglucosaminyltransferase2 (beta 1,4-) PON1 ENSG00000005421 Paraoxonase 1 PON2 ENSG00000105854Paraoxonase 2 PON3 ENSG00000105852 Paraoxonase 3 POSTN ENSG00000133110Periostin, osteoblast specific factor PPBP ENSG00000163736 Pro-plateletbasic protein (chemokine (C-X-C motif) ligand 7) PPIB ENSG00000166794Peptidylprolyl isomerase B (cyclophilin B) PPIC ENSG00000168938Peptidylprolyl isomerase C (cyclophilin C) PPDX ENSG00000143224Protoporphyrinogen oxidase PPP1CA ENSG00000172531 Protein phosphatase 1,catalytic subunit, alpha isozyme PPT1 ENSG00000131238 Palmitoyl-proteinthioesterase 1 PPT2 ENSG00000221988 Palmitoyl-protein thioesterase 2 PPYENSG00000108849 Pancreatic polypeptide PRAC2 ENSG00000229637 Prostatecancer susceptibility candidate 2 PRADC1 ENSG00000135617Protease-associated domain containing 1 PRAP1 ENSG00000165828Proline-rich acidic protein 1 PRB1 ENSG00000251655 Proline-rich proteinBstNI subfamily 1 PRB2 ENSG00000121335 Proline-rich protein BstNIsubfamily 2 PRB3 ENSG00000197870 Proline-rich protein BstNI subfamily 3PRB4 ENSG00000230657 Proline-rich protein BstNI subfamily 4 PRCDENSG00000214140 Progressive rod-cone degeneration PRCP ENSG00000137509Prolylcalboxypeptidase (angiotensinase C) PRDM12 ENSG00000130711 PRdomain containing 12 PRDX4 ENSG00000123131 Peroxiredoxin 4 PRELPENSG00000188783 Proline/arginine-rich end leucine-rich repeat proteinPRF1 ENSG00000180644 Perforin 1 (pore forming protein) PRG2ENSG00000186652 Proteoglycan 2, bone marrow (natural killer cellactivator, eosinophil granule major basic protein) PRG3 ENSG00000156575Proteoglycan 3 PRG4 ENSG00000116690 Proteoglycan 4 PRH1 ENSG00000231887Proline-rich protein HaeIII subfamily 1 PRH2 ENSG00000134551Proline-rich protein HaeIII subfamily 2 PRKAG1 ENSG00000181929 Proteinkinase, AMP-activated, gamma 1 non-catalytic subunit PRKCSHENSG00000130175 Protein kinase C substrate 80K-H PRKD1 ENSG00000184304Protein kinase D1 PRL ENSG00000172179 Prolactin PRLH ENSG00000071677Prolactin releasing hormone PRLR ENSG00000113494 Prolactin receptor PRNPENSG00000171867 Prion protein PRNT ENSG00000180259 Prion protein (testisspecific) PROC ENSG00000115718 Protein C (inactivator of coagulationfactors Va and VIIIa) PROK1 ENSG00000143125 Prokineticin 1 PROK2ENSG00000163421 Prokineticin 2 PROL1 ENSG00000171199 Proline rich,lacrimal 1 PROM1 ENSG00000007062 Prominin 1 PROS1 ENSG00000184500Protein S (alpha) PROZ ENSG00000126231 Protein Z, vitamin K-dependentplasma glycoprotein PRR27 ENSG00000187533 Proline rich 27 PRR4ENSG00000111215 Proline rich 4 (lacrimal) PRRG2 ENSG00000126460 Prolinerich Gla (G-carboxyglutamic acid) 2 PRRT3 ENSG00000163704 Proline-richtransmembrane protein 3 PRRT4 ENSG00000224940 Proline-rich transmembraneprotein 4 PRSS1 ENSG00000204983 Protease, serine, 1 (trypsin 1) PRSS12ENSG00000164099 Protease, serine, 12 (neurotrypsin, motopsin) PRSS16ENSG00000112812 Protease, serine, 16 (thymus) PRSS2 ENSG00000275896Protease, serine, 2 (trypsin 2) PRSS21 ENSG00000007038 Protease, serine,21 (testisin) PRSS22 ENSG00000005001 Protease, serine, 22 PRSS23ENSG00000150687 Protease, serine, 23 PRSS27 ENSG00000172382 Protease,serine 27 PRSS3 ENSG00000010438 Protease, serine, 3 PRSS33ENSG00000103355 Protease, serine, 33 PRSS35 ENSG00000146250 Protease,serine, 35 PRSS36 ENSG00000178226 Protease, serine, 36 PRSS37ENSG00000165076 Protease, serine, 37 PRSS38 ENSG00000185888 Protease,serine, 38 PRSS42 ENSG00000178055 Protease, serine, 42 PRSS48ENSG00000189099 Protease, serine, 48 PRSS50 ENSG00000206549 Protease,serine, 50 PRSS53 ENSG00000151006 Protease, serine, 53 PRSS54ENSG00000103023 Protease, serine, 54 PRSS55 ENSG00000184647 Protease,serine, 55 PRSS56 ENSG00000237412 Protease, serine, 56 PRSS57ENSG00000185198 Protease, serine, 57 PRSS58 ENSG00000258223 Protease,serine, 58 PRSS8 ENSG00000052344 Protease, serine, 8 PRTGENSG00000166450 Protogenin PRTN3 ENSG00000196415 Proteinase 3 PSAPENSG00000197746 Prosaposin PSAPL1 ENSG00000178597 Prosaposin-like 1(gene/pseudogene) PSG1 ENSG00000231924 Pregnancy specificbeta-1-glycoprotein 1 PSG11 ENSG00000243130 Pregnancy specificbeta-1-glycoprotein 11 PSG2 ENSG00000242221 Pregnancy specificbeta-1-glycoprotein 2 PSG3 ENSG00000221826 Pregnancy specificbeta-1-glycoprotein 3 PSG4 ENSG00000243137 Pregnancy specificbeta-1-glycoprotein 4 PSG5 ENSG00000204941 Pregnancy specificbeta-1-glycoprotein 5 PSG6 ENSG00000170848 Pregnancy specificbeta-1-glycoprotein 6 PSG7 ENSG00000221878 Pregnancy specificbeta-1-glycoprotein 7 (gene/pseudogene) PSG8 ENSG00000124467 Pregnancyspecific beta-1-glycoprotein 8 PSG9 ENSG00000183668 Pregnancy specificbeta-1-glycoprotein 9 PSMD1 ENSG00000173692 Proteasome 26S subunit,non-ATPase 1 PSORS1C2 ENSG00000204538 Psoriasis susceptibility 1candidate 2 PSPN ENSG00000125650 Persephin PTGDS ENSG00000107317Prostaglandin D2 synthase 21 kDa (brain) PTGIR ENSG00000160013Prostaglandin 12 (prostacyclin) receptor (IP) PTGS1 ENSG00000095303Prostaglandin-endoperoxide synthase 1 (prostaglandin G/H synthase andcyclooxygenase) PTGS2 ENSG00000073756 Prostaglandin-endoperoxidesynthase 2 (prostaglandin G/H synthase and cyclooxygenase) PTHENSG00000152266 Parathyroid hormone PTH2 ENSG00000142538 Parathyroidhormone 2 PTHLH ENSG00000087494 Parathyroid hormone-like hormone PTK7ENSG00000112655 Protein tyrosine kinase 7 (inactive) PTN ENSG00000105894Pleiotrophin PTPRA ENSG00000132670 Protein tyrosine phosphatase,receptor type, A PTPRB ENSG00000127329 Protein tyrosine phosphatase,receptor type, B PTPRC ENSG00000081237 Protein tyrosine phosphatase,receptor type, C PTPRCAP ENSG00000213402 Protein tyrosine phosphatase,receptor type, C-associated protein PTPRD ENSG00000153707 Proteintyrosine phosphatase, receptor type, D PTPRF ENSG00000142949 Proteintyrosine phosphatase, receptor type, F PTPRJ ENSG00000149177 Proteintyrosine phosphatase, receptor type, J PTPRO ENSG00000151490 Proteintyrosine phosphatase, receptor type, O PTPRS ENSG00000105426 Proteintyrosine phosphatase, receptor type, S PTTG1IP ENSG00000183255 Pituitarytumor-transforming 1 interacting protein PTX3 ENSG00000163661 Pentraxin3, long PTX4 ENSG00000251692 Pentraxin 4, long PVR ENSG00000073008Poliovirus receptor PVRL1 ENSG00000110400 Poliovirus receptor-related 1(herpesvirus entry mediator C) PXDN ENSG00000130508 Peroxidasin PXDNLENSG00000147485 Peroxidasin-like PXYLP1 ENSG00000155893 2-phosphoxylosephosphatase 1 PYY ENSG00000131096 Peptide YY PZP ENSG00000126838Pregnancy-zone protein QPCT ENSG00000115828 Glutaminyl-peptidecyclotransferase QPRT ENSG00000103485 Quinolinatephosphoribosyltransferase QRFP ENSG00000188710 Pyroglutamylated RFamidepeptide QS0X1 ENSG00000116260 Quiescin Q6 sulfhydryl oxidase 1 R3HDMLENSG00000101074 R3H domain containing-like RAB26 ENSG00000167964 RAB26,member RAS oncogene family RAB36 ENSG00000100228 RAB36, member RASoncogene family RAB9B ENSG00000123570 RAB9B, member RAS oncogene familyRAET1E ENSG00000164520 Retinoic acid early transcript 1E RAET1GENSG00000203722 Retinoic acid early transcript 1G RAMP2 ENSG00000131477Receptor (G protein-coupled) activity modifying protein 2 RAPGEF5ENSG00000136237 Rap guanine nucleotide exchange factor (GEF) 5 RARRES1ENSG00000118849 Retinoic acid receptor responder (tazarotene induced) 1RARRES2 ENSG00000106538 Retinoic acid receptor responder (tazaroteneinduced) 2 RASA2 ENSG00000155903 RAS p21 protein activator 2 RBM3ENSG00000102317 RNA binding motif (RNP1, RRM) protein 3 RBP3ENSG00000265203 Retinol binding protein 3, interstitial RBP4ENSG00000138207 Retinol binding protein 4, plasma RCN1 ENSG00000049449Reticulocalbin 1, EF-hand calcium binding domain RCN2 ENSG00000117906Reticulocalbin 2, EF-hand calcium binding domain RCN3 ENSG00000142552Reticulocalbin 3, EF-hand calcium binding domain RCOR1 ENSG00000089902REST corepressor 1 RDH11 ENSG00000072042 Retinol dehydrogenase 11(all-trans/9-cis/11-cis) RDH12 ENSG00000139988 Retinol dehydrogenase 12(all-trans/9-cis/11-cis) RDH13 ENSG00000160439 Retinol dehydrogenase 13(all-trans/9-cis) RDH5 ENSG00000135437 Retinol dehydrogenase 5(11-cis/9-cis) RDH8 ENSG00000080511 Retinol dehydrogenase 8 (all-trans)REG1A ENSG00000115386 Regenerating islet-derived 1 alpha REG1BENSG00000172023 Regenerating islet-derived 1 beta REG3A ENSG00000172016Regenerating islet-derived 3 alpha REG3G ENSG00000143954 Regeneratingislet-derived 3 gamma REG4 ENSG00000134193 Regenerating islet-derivedfamily, member 4 RELN ENSG00000189056 Reelin RELT ENSG00000054967 RELTtumor necrosis factor receptor REN ENSG00000143839 Renin REPIN1ENSG00000214022 Replication initiator 1 REPS2 ENSG00000169891 RALBP1associated Eps domain containing 2 RET ENSG00000165731 Retproto-oncogene RETN ENSG00000104918 Resistin RETNLB ENSG00000163515Resistin like beta RETSAT ENSG00000042445 Retinol saturase(all-trans-retinol 13,14-reductase) RFNG ENSG00000169733 RFNGO-fucosylpeptide 3-beta-N-acetylglucosaminyltransferase RGCCENSG00000102760 Regulator of cell cycle RGL4 ENSG00000159496 Ral guaninenucleotide dissociation stimulator-like 4 RGMA ENSG00000182175 Repulsiveguidance molecule family member a RGMB ENSG00000174136 Repulsiveguidance molecule family member b RHOQ ENSG00000119729 Ras homologfamily member Q RIC3 ENSG00000166405 RIC3 acetylcholine receptorchaperone RIMS1 ENSG00000079841 Regulating synaptic membrane exocytosis1 RIPPLY1 ENSG00000147223 Ripply transcriptional repressor 1 RLN1ENSG00000107018 Relaxin 1 RLN2 ENSG00000107014 Relaxin 2 RLN3ENSG00000171136 Relaxin 3 RMDN1 ENSG00000176623 Regulator of microtubuledynamics 1 RNASE1 ENSG00000129538 Ribonuclease, RNase A family, 1(pancreatic) RNASE10 ENSG00000182545 Ribonuclease, RNase A family, 10(non-active) RNASEll ENSG00000173464 Ribonuclease, RNase A family, 11(non-active) RNASE12 ENSG00000258436 Ribonuclease, RNase A family, 12(non-active) RNASE13 ENSG00000206150 Ribonuclease, RNase A family, 13(non-active) RNASE2 ENSG00000169385 Ribonuclease, RNase A family, 2(liver, eosinophil-derived neurotoxin) RNASE3 ENSG00000169397Ribonuclease, RNase A family, 3 RNASE4 ENSG00000258818 Ribonuclease,RNase A family, 4 RNASE6 ENSG00000169413 Ribonuclease, RNase A family,k6 RNASE7 ENSG00000165799 Ribonuclease, RNase A family, 7 RNASE8ENSG00000173431 Ribonuclease, RNase A family, 8 RNASE9 ENSG00000188655Ribonuclease, RNase A family, 9 (non-active) RNASEH1 ENSG00000171865Ribonuclease H1 RNASET2 ENSG00000026297 Ribonuclease T2 RNF146ENSG00000118518 Ring finger protein 146 RNF148 ENSG00000235631 Ringfinger protein 148 RNF150 ENSG00000170153 Ring finger protein 150 RNF167ENSG00000108523 Ring finger protein 167 RNF220 ENSG00000187147 Ringfinger protein 220 RNF34 ENSG00000170633 Ring finger protein 34, E3ubiquitin protein ligase RNLS ENSG00000184719 Renalase, FAD-dependentamine oxidase RNPEP ENSG00000176393 Arginyl aminopeptidase(aminopeptidase B) ROR1 ENSG00000185483 Receptor tyrosine kinase-likeorphan receptor 1 RP11-1236K1.1 ENSG00000233050 RP11-14J7.7ENSG00000259060 RP11-196G11.1 ENSG00000255439 RP11-350014.18ENSG00000261793 RP11-520P18.5 ENSG00000261667 RP11-812E19.9ENSG00000259680 RP11-903H12.5 ENSG00000259171 RP11-977G19.10ENSG00000144785 RP4-576H24.4 ENSG00000260861 RP4-608015.3ENSG00000276911 Complement factor H-related protein 2 RPL3ENSG00000100316 Ribosomal protein L3 RPLP2 ENSG00000177600 Ribosomalprotein, large, P2 RPN2 ENSG00000118705 Ribophorin II RPS27LENSG00000185088 Ribosomal protein S27-like RQCD1 ENSG00000144580 RCD1required for cell differentiationl homolog (S. pombe) RS1ENSG00000102104 Retinoschisin 1 RSF1 ENSG00000048649 Remodeling andspacing factor 1 RSPO1 ENSG00000169218 R-spondin 1 RSPO2 ENSG00000147655R-spondin 2 RSPO3 ENSG00000146374 R-spondin 3 RSPO4 ENSG00000101282R-spondin 4 RSPRY1 ENSG00000159579 Ring finger and SPRY domaincontaining 1 RTBDN ENSG00000132026 Retbindin RTN4RL1 ENSG00000185924Reticulon 4 receptor-like 1 RTN4RL2 ENSG00000186907 Reticulon 4receptor-like 2 SAA1 ENSG00000173432 Serum amyloid A1 SAA2ENSG00000134339 Serum amyloid A2 SAA4 ENSG00000148965 Serum amyloid A4,constitutive SAP30 ENSG00000164105 Sin3A-associated protein, 30 kDaSAR1A ENSG00000079332 Secretion associated, Ras related GTPase 1A SARAFENSG00000133872 Store-operated calcium entry-associated regulatoryfactor SARM1 ENSG00000004139 Sterile alpha and TIR motif containing 1SATB1 ENSG00000182568 SATB homeobox 1 SAXO2 ENSG00000188659 Stabilizerof axonemal microtubules 2 SBSN ENSG00000189001 Suprabasin SBSPONENSG00000164764 Somatomedin B and thrombospondin, type 1 domaincontaining SCARF1 ENSG00000074660 Scavenger receptor class F, member 1SCG2 ENSG00000171951 Secretogranin II SCG3 ENSG00000104112 SecretograninIII SCG5 ENSG00000166922 Secretogranin V SCGB1A1 ENSG00000149021Secretoglobin, family 1A, member 1 (uteroglobin) SCGB1C1 ENSG00000188076Secretoglobin, family 1C, member 1 SCGB1C2 ENSG00000268320Secretoglobin, family 1C, member 2 SCGB1D1 ENSG00000168515Secretoglobin, family 1D, member 1 SCGB1D2 ENSG00000124935Secretoglobin, family 1D, member 2 SCGB1D4 ENSG00000197745Secretoglobin, family 1D, member 4 SCGB2A1 ENSG00000124939Secretoglobin, family 2A, member 1 SCGB2A2 ENSG00000110484Secretoglobin, family 2A, member 2 SCGB2B2 ENSG00000205209Secretoglobin, family 2B, member 2 SCGB3A1 ENSG00000161055Secretoglobin, family 3A, member 1 SCGB3A2 ENSG00000164265Secretoglobin, family 3A, member 2 SCN1B ENSG00000105711 Sodium channel,voltage gated, type I beta subunit SCN3B ENSG00000166257 Sodium channel,voltage gated, type III beta subunit SCPEP1 ENSG00000121064 Serinecalboxypeptidase 1 SCRG1 ENSG00000164106 Stimulator of chondrogenesis 1SCT ENSG00000070031 Secretin SCUBE1 ENSG00000159307 Signal peptide, CUBdomain, EGF-like 1 SCUBE2 ENSG00000175356 Signal peptide, CUB domain,EGF-like 2 SCUBE3 ENSG00000146197 Signal peptide, CUB domain, EGF-like 3SDC1 ENSG00000115884 Syndecan 1 SDF2 ENSG00000132581 Stromalcell-derived factor 2 SDF2L1 ENSG00000128228 Stromal cell-derived factor2-like 1 SDF4 ENSG00000078808 Stromal cell derived factor 4 SDHAF2ENSG00000167985 Succinate dehydrogenase complex assembly factor 2 SDHAF4ENSG00000154079 Succinate dehydrogenase complex assembly factor 4 SDHBENSG00000117118 Succinate dehydrogenase complex, subunit B, iron sulfur(Ip) SDHD ENSG00000204370 Succinate dehydrogenase complex, subunit D,integral membrane protein SEC14L3 ENSG00000100012 SEC14-like lipidbinding 3 SEC16A ENSG00000148396 SEC16 homolog A, endoplasmic reticulumexport factor SEC16B ENSG00000120341 SEC16 homolog B, endoplasmicreticulum export factor SEC22C ENSG00000093183 SEC22 homolog C, vesicletrafficking protein SEC31A ENSG00000138674 SEC31 homolog A, COPII coatcomplex component SECISBP2 ENSG00000187742 SECIS binding protein 2SECTM1 ENSG00000141574 Secreted and transmembrane 1 SEL1LENSG00000071537 Sel-1 suppressor of lin-12-like (C. elegans) SELMENSG00000198832 Selenoprotein M SELO ENSG00000073169 Selenoprotein OSEMA3A ENSG00000075213 Sema domain, immunoglobulin domain (Ig), shortbasic domain, secreted, (semaphorin) 3A SEMA3B ENSG00000012171 Semadomain, immunoglobulin domain (Ig), short basic domain, secreted,(semaphorin) 3B SEMA3C ENSG00000075223 Sema domain, immunoglobulindomain (Ig), short basic domain, secreted, (semaphorin) 3C SEMA3EENSG00000170381 Sema domain, immunoglobulin domain (Ig), short basicdomain, secreted, (semaphorin) 3E SEMA3F ENSG00000001617 Sema domain,immunoglobulin domain (Ig), short basic domain, secreted, (semaphorin)3F SEMA3G ENSG00000010319 Sema domain, immunoglobulin domain (Ig), shortbasic domain, secreted, (semaphorin) 3G SEMA4A ENSG00000196189 Semadomain, immunoglobulin domain (Ig), transmembrane domain (TM) and shortcytoplasmic domain, (semaphorin) 4A SEMA4B ENSG00000185033 Sema domain,immunoglobulin domain (Ig), transmembrane domain (TM) and shortcytoplasmic domain, (semaphorin) 4B SEMA4C ENSG00000168758 Sema domain,immunoglobulin domain (Ig), transmembrane domain (TM) and shortcytoplasmic domain, (semaphorin) 4C SEMA4D ENSG00000187764 Sema domain,immunoglobulin domain (Ig), transmembrane domain (TM) and shortcytoplasmic domain, (semaphorin) 4D SEMA4F ENSG00000135622 Sema domain,immunoglobulin domain (Ig), transmembrane domain (TM) and shortcytoplasmic domain, (semaphorin) 4F SEMA4G ENSG00000095539 Sema domain,immunoglobulin domain (Ig), transmembrane domain (TM) and shortcytoplasmic domain, (semaphorin) 4G SEMA5A ENSG00000112902 Sema domain,seven thrombospondin repeats (type 1 and type 1- like), transmembranedomain (TM) and short cytoplasmic domain, (semaphorin) 5A SEMA6AENSG00000092421 Sema domain, transmembrane domain (TM), and cytoplasmicdomain, (semaphorin) 6A SEMA6C ENSG00000143434 Sema domain,transmembrane domain (TM), and cytoplasmic domain, (semaphorin) 6CSEMA6D ENSG00000137872 Sema domain, transmembrane domain (TM), andcytoplasmic domain, (semaphorin) 6D SEMG1 ENSG00000124233 Semenogelin ISEMG2 ENSG00000124157 Semenogelin II 15-Sep ENSG00000183291 15 kDaselenoprotein SEPN1 ENSG00000162430 Selenoprotein N, 1 SEPP1ENSG00000250722 Selenoprotein P, plasma, 1 9-Sep ENSG00000184640 Septin9 SERPINA1 ENSG00000197249 Serpin peptidase inhibitor, clade A (alpha-1antiproteinase, antitrypsin), member 1 SERPINA10 ENSG00000140093 Serpinpeptidase inhibitor, clade A (alpha-1 antiproteinase, antitrypsin),member 10 SERPINA11 ENSG00000186910 Serpin peptidase inhibitor, clade A(alpha-1 antiproteinase, antitrypsin), member 11 SERPINA12ENSG00000165953 Serpin peptidase inhibitor, clade A (alpha-1antiproteinase, antitrypsin), member 12 SERPINA3 ENSG00000196136 Serpinpeptidase inhibitor, clade A (alpha-1 antiproteinase, antitrypsin),member 3 SERPINA3 ENSG00000273259 Serpin peptidase inhibitor, clade A(alpha-1 antiproteinase, antitrypsin), member 3 SERPINA4 ENSG00000100665Serpin peptidase inhibitor, clade A (alpha-1 antiproteinase,antitrypsin), member 4 SERPINA5 ENSG00000188488 Serpin peptidaseinhibitor, clade A (alpha-1 antiproteinase, antitrypsin), member 5SERPINA6 ENSG00000170099 Serpin peptidase inhibitor, clade A (alpha-1antiproteinase, antitrypsin), member 6 SERPINA7 ENSG00000123561 Serpinpeptidase inhibitor, clade A (alpha-1 antiproteinase, antitrypsin),member 7 SERPINA9 ENSG00000170054 Serpin peptidase inhibitor, clade A(alpha-1 antiproteinase, antitrypsin), member 9 SERPINB2 ENSG00000197632Serpin peptidase inhibitor, clade B (ovalbumin), member 2 SERPINC1ENSG00000117601 Serpin peptidase inhibitor, clade C (antithrombin),member 1 SERPIND1 ENSG00000099937 Serpin peptidase inhibitor, clade D(heparin cofactor), member 1 SERPINE1 ENSG00000106366 Serpin peptidaseinhibitor, clade E (nexin, plasminogen activator inhibitor type 1),member 1 SERPINE2 ENSG00000135919 Serpin peptidase inhibitor, clade E(nexin, plasminogen activator inhibitor type 1), member 2 SERPINE3ENSG00000253309 Serpin peptidase inhibitor, clade E (nexin, plasminogenactivator inhibitor type 1), member 3 SERPINF1 ENSG00000132386 Serpinpeptidase inhibitor, clade F (alpha-2 antiplasmin, pigment epitheliumderived factor), member 1 SERPINF2 ENSG00000167711 Serpin peptidaseinhibitor, clade F (alpha-2 antiplasmin, pigment epithelium derivedfactor), member 2 SERPING1 ENSG00000149131 Serpin peptidase inhibitor,clade G (C1 inhibitor), member 1 SERPINH1 ENSG00000149257 Serpinpeptidase inhibitor, clade H (heat shock protein 47), member 1,(collagen binding protein 1) SERPINI1 ENSG00000163536 Serpin peptidaseinhibitor, clade I (neuroserpin), member 1 SERPINI2 ENSG00000114204Serpin peptidase inhibitor, clade I (pancpin), member 2 SETD8ENSG00000183955 SET domain containing (lysine methyltransferase) 8SEZ6L2 ENSG00000174938 Seizure related 6 homolog (mouse)-like 2 SFRP1ENSG00000104332 Secreted frizzled-related protein 1 SFRP2ENSG00000145423 Secreted frizzled-related protein 2 SFRP4ENSG00000106483 Secreted frizzled-related protein 4 SFRP5ENSG00000120057 Secreted frizzled-related protein 5 SFTA2ENSG00000196260 Surfactant associated 2 SFTPA1 ENSG00000122852Surfactant protein Al SFTPA2 ENSG00000185303 Surfactant protein A2 SFTPBENSG00000168878 Surfactant protein B SFTPD ENSG00000133661 Surfactantprotein D SFXN5 ENSG00000144040 Sideroflexin 5 SGCA ENSG00000108823Sarcoglycan, alpha (50 kDa dystrophin-associated glycoprotein) SGSHENSG00000181523 N-sulfoglucosamine sulfohydrolase SH3RF3 ENSG00000172985SH3 domain containing ring finger 3 SHBG ENSG00000129214 Sexhormone-binding globulin SHE ENSG00000169291 Src homology 2 domaincontaining E SHH ENSG00000164690 Sonic hedgehog SHKBP1 ENSG00000160410SH3KBP1 binding protein 1 SIAE ENSG00000110013 Sialic acidacetylesterase SIDT2 ENSG00000149577 SIDI_ transmembrane family, member2 SIGLEC10 ENSG00000142512 Sialic acid binding Ig-like lectin 10 SIGLEC6ENSG00000105492 Sialic acid binding Ig-like lectin 6 SIGLEC7ENSG00000168995 Sialic acid binding Ig-like lectin 7 SIGLECL1ENSG00000179213 SIGLEC family like 1 SIGMAR1 ENSG00000147955 Sigmanon-opioid intracellular receptor 1 SIL1 ENSG00000120725 SIL1 nucleotideexchange factor SIRPB1 ENSG00000101307 Signal-regulatory protein beta 1SIRPD ENSG00000125900 Signal-regulatory protein delta SLAMF1ENSG00000117090 Signaling lymphocytic activation molecule family member1 SLAMF7 ENSG00000026751 SLAM family member 7 SLC10A3 ENSG00000126903Solute carrier family 10, member 3 SLC15A3 ENSG00000110446 Solutecarrier family 15 (oligopeptide transporter), member 3 SLC25A14ENSG00000102078 Solute carrier family 25 (mitochondrial carrier, brain),member 14 SLC25A25 ENSG00000148339 Solute carrier family 25(mitochondrial carrier; phosphate carrier), member 25 SLC2A5ENSG00000142583 Solute carrier family 2 (facilitated glucose/fructosetransporter), member 5 SLC35E3 ENSG00000175782 Solute carrier family 35,member E3 SLC39A10 ENSG00000196950 Solute carrier family 39 (zinctransporter), member 10 SLC39A14 ENSG00000104635 Solute carrier family39 (zinc transporter), member 14 SLC39A4 ENSG00000147804 Solute carrierfamily 39 (zinc transporter), member 4 SLC39A5 ENSG00000139540 Solutecarrier family 39 (zinc transporter), member 5 SLC3A1 ENSG00000138079Solute carrier family 3 (amino acid transporter heavy chain), member 1SLC51A ENSG00000163959 Solute carrier family 51, alpha subunit SLC52A2ENSG00000185803 Solute carrier family 52 (riboflavin transporter),member 2 SLC5A6 ENSG00000138074 Solute carrier family 5(sodium/multivitamin and iodide cotransporter), member 6 SLC6A9ENSG00000196517 Solute carrier family 6 (neurotransmitter transporter,glycine), member 9 SLC8A1 ENSG00000183023 Solute carrier family 8(sodium/calcium exchanger), member 1 SLC8B1 ENSG00000089060 Solutecarrier family 8 (sodium/lithium/calcium exchanger), member B1 SLC9A6ENSG00000198689 Solute carrier family 9, subfamily A (NHE6, cationproton antiporter 6), member 6 SLCO1A2 ENSG00000084453 Solute carrierorganic anion transporter family, member 1A2 SLIT1 ENSG00000187122 Slitguidance ligand 1 SLIT2 ENSG00000145147 Slit guidance ligand 2 SLIT3ENSG00000184347 Slit guidance ligand 3 SLITRK3 ENSG00000121871 SLIT andNTRK-like family, member 3 SLPI ENSG00000124107 Secretory leukocytepeptidase inhibitor SLTM ENSG00000137776 SAFB-like, transcriptionmodulator SLURP1 ENSG00000126233 Secreted LY6/PLAUR domain containing 1SMARCA2 ENSG00000080503 SWI/SNF related, matrix associated, actindependent regulator of chromatin, subfamily a, member 2 SMG6ENSG00000070366 SMG6 nonsense mediated mRNA decay factor SMIM7ENSG00000214046 Small integral membrane protein 7 SMOC1 ENSG00000198732SPARC related modular calcium binding 1 SMOC2 ENSG00000112562 SPARCrelated modular calcium binding 2 SMPDL3A ENSG00000172594 Sphingomyelinphosphodiesterase, acid-like 3A SMPDL3B ENSG00000130768 Sphingomyelinphosphodiesterase, acid-like 3B SMR3A ENSG00000109208 Submaxillary glandandrogen regulated protein 3A SMR3B ENSG00000171201 Submaxillary glandandrogen regulated protein 3B SNED1 ENSG00000162804 Sushi, nidogen andEGF-like domains 1 SNTB1 ENSG00000172164 Syntrophin, beta 1(dystrophin-associated protein A1, 59 kDa, basic component 1) SNTB2ENSG00000168807 Syntrophin, beta 2 (dystrophin-associated protein A1, 59kDa, basic component 2) SNX14 ENSG00000135317 Sorting nexin 14 SOD3ENSG00000109610 Superoxide dismutase 3, extracellular SOSTENSG00000167941 Sclerostin SOSTDC1 ENSG00000171243 Sclerostin domaincontaining 1 SOWAHA ENSG00000198944 Sosondowah ankyrin repeat domainfamily member A SPACA3 ENSG00000141316 Sperm acrosome associated 3SPACA4 ENSG00000177202 Sperm acrosome associated 4 SPACA5ENSG00000171489 Sperm acrosome associated 5 SPACA5B ENSG00000171478Sperm acrosome associated 5B SPACA7 ENSG00000153498 Sperm acrosomeassociated 7 SPAG11A ENSG00000178287 Sperm associated antigen 11ASPAG11B ENSG00000164871 Sperm associated antigen 11B SPARCENSG00000113140 Secreted protein, acidic, cysteine-rich (osteonectin)SPARCL1 ENSG00000152583 SPARC-like 1 (hevin) SPATA20 ENSG00000006282Spermatogenesis associated 20 SPESP1 ENSG00000258484 Sperm equatorialsegment protein 1 SPINK1 ENSG00000164266 Serine peptidase inhibitor,Kazal type 1 SPINK13 ENSG00000214510 Serine peptidase inhibitor, Kazaltype 13 (putative) SPINK14 ENSG00000196800 Serine peptidase inhibitor,Kazal type 14 (putative) SPINK2 ENSG00000128040 Serine peptidaseinhibitor, Kazal type 2 (acrosin-trypsin inhibitor) SPINK4ENSG00000122711 Serine peptidase inhibitor, Kazal type 4 SPINK5ENSG00000133710 Serine peptidase inhibitor, Kazal type 5 SPINK6ENSG00000178172 Serine peptidase inhibitor, Kazal type 6 SPINK7ENSG00000145879 Serine peptidase inhibitor, Kazal type 7 (putative)SPINK8 ENSG00000229453 Serine peptidase inhibitor, Kazal type 8(putative) SPINK9 ENSG00000204909 Serine peptidase inhibitor, Kazal type9 SPINT1 ENSG00000166145 Serine peptidase inhibitor, Kunitz type 1SPINT2 ENSG00000167642 Serine peptidase inhibitor, Kunitz type, 2 SPINT3ENSG00000101446 Serine peptidase inhibitor, Kunitz type, 3 SPINT4ENSG00000149651 Serine peptidase inhibitor, Kunitz type 4 SPOCK1ENSG00000152377 Sparc/osteonectin, cwcv and kazal-like domainsproteoglycan (testican) 1 SPOCK2 ENSG00000107742 Sparc/osteonectin, cwcvand kazal-like domains proteoglycan (testican) 2 SPOCK3 ENSG00000196104Sparc/osteonectin, cwcv and kazal-like domains proteoglycan (testican) 3SPON1 ENSG00000262655 Spondin 1, extracellular matrix protein SPON2ENSG00000159674 Spondin 2, extracellular matrix protein SPP1ENSG00000118785 Secreted phosphoprotein 1 SPP2 ENSG00000072080 Secretedphosphoprotein 2, 24 kDa SPRN ENSG00000203772 Shadow of prion proteinhomolog (zebrafish) SPRYD3 ENSG00000167778 SPRY domain containing 3SPRYD4 ENSG00000176422 SPRY domain containing 4 SPTY2D1-AS1ENSG00000247595 SPTY2D1 antisense RNA 1 SPX ENSG00000134548 Spexinhormone SRGN ENSG00000122862 Serglycin SRL ENSG00000185739 SarcalumeninSRP14 ENSG00000140319 Signal recognition particle 14 kDa (homologous AluRNA binding protein) SRPX ENSG00000101955 Sushi-repeat containingprotein, X-linked SRPX2 ENSG00000102359 Sushi-repeat containing protein,X-linked 2 SSC4D ENSG00000146700 Scavenger receptor cysteine richfamily, 4 domains SSC5D ENSG00000179954 Scavenger receptor cysteine richfamily, 5 domains SSPO ENSG00000197558 SCO-spondin SSR2 ENSG00000163479Signal sequence receptor, beta (translocon-associated protein beta) SSTENSG00000157005 Somatostatin ST3GAL1 ENSG00000008513 ST3beta-galactoside alpha-2,3-sialyltransferase 1 ST3GAL4 ENSG00000110080ST3 beta-galactoside alpha-2,3-sialyltransferase 4 ST6GAL1ENSG00000073849 ST6 beta-galactosamide alpha-2,6-sialyltranferase 1ST6GALNAC2 ENSG00000070731 ST6(alpha-N-acetyl-neuraminyl-2,3-beta-galactosyl-1,3)-N-acetylgalactosaminide alpha-2,6-sialyltransferase 2 ST6GALNAC5ENSG00000117069 ST6(alpha-N-acetyl-neuraminyl-2,3-beta-galactosyl-1,3)-N-acetylgalactosaminide alpha-2,6-sialyltransferase 5 ST6GALNAC6ENSG00000160408 ST6(alpha-N-acetyl-neuraminyl-2,3-beta-galactosyl-1,3)-N-acetylgalactosaminide alpha-2,6-sialyltransferase 6 ST8SIA2ENSG00000140557 ST8 alpha-N-acetyl-neuraminidealpha-2,8-sialyltransferase 2 ST8SIA4 ENSG00000113532 ST8alpha-N-acetyl-neuraminide alpha-2,8-sialyltransferase 4 ST8SIA6ENSG00000148488 ST8 alpha-N-acetyl-neuraminidealpha-2,8-sialyltransferase 6 STARD7 ENSG00000084090 StAR-related lipidtransfer (START) domain containing 7 STATH ENSG00000126549 StatherinSTC1 ENSG00000159167 Stanniocalcin 1 STC2 ENSG00000113739 Stanniocalcin2 STMND1 ENSG00000230873 Stathmin domain containing 1 STOML2ENSG00000165283 Stomatin (EPB72)-like 2 STOX1 ENSG00000165730 Storkheadbox 1 STRC ENSG00000242866 Stereocilin SUCLG1 ENSG00000163541Succinate-CoA ligase, alpha subunit SUDS3 ENSG00000111707 SDS3 homolog,SIN3A corepressor complex component SULF1 ENSG00000137573 Sulfatase 1SULF2 ENSG00000196562 Sulfatase 2 SUMF1 ENSG00000144455 Sulfatasemodifying factor 1 SUMF2 ENSG00000129103 Sulfatase modifying factor 2SUSD1 ENSG00000106868 Sushi domain containing 1 SUSD5 ENSG00000173705Sushi domain containing 5 SVEP1 ENSG00000165124 Sushi, von Willebrandfactor type A, EGF and pentraxin domain containing 1 SWSAP1ENSG00000173928 SWIM-type zinc finger 7 associated protein 1 SYAP1ENSG00000169895 Synapse associated protein 1 SYCN ENSG00000179751Syncollin TAC1 ENSG00000006128 Tachykinin, precursor 1 TAC3ENSG00000166863 Tachykinin 3 TAC4 ENSG00000176358 Tachykinin 4(hemokinin) TAGLN2 ENSG00000158710 Transgelin 2 TAPBP ENSG00000231925TAP binding protein (tapasin) TAPBPL ENSG00000139192 TAP bindingprotein-like TBL2 ENSG00000106638 Transducin (beta)-like 2 TBX10ENSG00000167800 T-box 10 TCF12 ENSG00000140262 Transcription factor 12TCN1 ENSG00000134827 Transcobalamin I (vitamin B12 binding protein, Rbinder family) TCN2 ENSG00000185339 Transcobalamin II TCTN1ENSG00000204852 Tectonic family member 1 TCTN3 ENSG00000119977 Tectonicfamily member 3 TDP2 ENSG00000111802 Tyrosyl-DNA phosphodiesterase 2 TEKENSG00000120156 ILK tyrosine kinase, endothelial TEPP ENSG00000159648Testis, prostate and placenta expressed TEX101 ENSG00000131126 Testisexpressed 101 TEX264 ENSG00000164081 Testis expressed 264 TFENSG00000091513 Transferrin TFAM ENSG00000108064 Transcription factor A,mitochondrial TFF1 ENSG00000160182 Trefoil factor 1 TFF2 ENSG00000160181Trefoil factor 2 TFF3 ENSG00000160180 Trefoil factor 3 (intestinal) TFPIENSG00000003436 Tissue factor pathway inhibitor (lipoprotein-associatedcoagulation inhibitor) TFPI2 ENSG00000105825 Tissue factor pathwayinhibitor 2 TG ENSG00000042832 Thyroglobulin TGFB1 ENSG00000105329Transforming growth factor, beta 1 TGFB2 ENSG00000092969 Transforminggrowth factor, beta 2 TGFB3 ENSG00000119699 Transforming growth factor,beta 3 TGFBI ENSG00000120708 Transforming growth factor, beta-induced,68 kDa TGFBR1 ENSG00000106799 Transforming growth factor, beta receptor1 TGFBR3 ENSG00000069702 Transforming growth factor, beta receptor IIITHBS1 ENSG00000137801 Thrombospondin 1 THBS2 ENSG00000186340Thrombospondin 2 THBS3 ENSG00000169231 Thrombospondin 3 THBS4ENSG00000113296 Thrombospondin 4 THOC3 ENSG00000051596 THO complex 3THPO ENSG00000090534 Thrombopoietin THSD4 ENSG00000187720Thrombospondin, type I, domain containing 4 THY1 ENSG00000154096 Thy-1cell surface antigen TIE1 ENSG00000066056 Tyrosine kinase withimmunoglobulin-like and EGF-like domains 1 TIMMDC1 ENSG00000113845Translocase of inner mitochondrial membrane domain containing 1 TIMP1ENSG00000102265 TIMP metallopeptidase inhibitor 1 TIMP2 ENSG00000035862TIMP metallopeptidase inhibitor 2 TIMP3 ENSG00000100234 TIMPmetallopeptidase inhibitor 3 TIMP4 ENSG00000157150 TIMP metallopeptidaseinhibitor 4 TINAGL1 ENSG00000142910 Tubulointerstitial nephritisantigen-like 1 TINF2 ENSG00000092330 TERF1 (TRF1)-interacting nuclearfactor 2 TLL2 ENSG00000095587 Tolloid-like 2 TLR1 ENSG00000174125Toll-like receptor 1 TLR3 ENSG00000164342 Toll-like receptor 3 TM2D2ENSG00000169490 TM2 domain containing 2 TM2D3 ENSG00000184277 TM2 domaincontaining 3 TM7SF3 ENSG00000064115 Transmembrane 7 superfamily member 3TM9SF1 ENSG00000100926 Transmembrane 9 superfamily member 1 TMC06ENSG00000113119 Transmembrane and coiled-coil domains 6 TMED1ENSG00000099203 Transmembrane p24 trafficking protein 1 TMED2ENSG00000086598 Transmembrane p24 trafficking protein 2 TMED3ENSG00000166557 Transmembrane p24 trafficking protein 3 TMED4ENSG00000158604 Transmembrane p24 trafficking protein 4 TMED5ENSG00000117500 Transmembrane p24 trafficking protein 5 TMED7ENSG00000134970 Transmembrane p24 trafficking protein 7 TMED7-TICAM2ENSG00000251201 TMED7-TICAM2 readthrough TMEM108 ENSG00000144868Transmembrane protein 108 TMEM116 ENSG00000198270 Transmembrane protein116 TMEM119 ENSG00000183160 Transmembrane protein 119 TMEM155ENSG00000164112 Transmembrane protein 155 TMEM168 ENSG00000146802Transmembrane protein 168 TMEM178A ENSG00000152154 Transmembrane protein178A TMEM179 ENSG00000258986 Transmembrane protein 179 TMEM196ENSG00000173452 Transmembrane protein 196 TMEM199 ENSG00000244045Transmembrane protein 199 TMEM205 ENSG00000105518 Transmembrane protein205 TMEM213 ENSG00000214128 Transmembrane protein 213 TMEM25ENSG00000149582 Transmembrane protein 25 TMEM30C ENSG00000235156Transmembrane protein 30C TMEM38B ENSG00000095209 Transmembrane protein38B TMEM44 ENSG00000145014 Transmembrane protein 44 TMEM52ENSG00000178821 Transmembrane protein 52 TMEM52B ENSG00000165685Transmembrane protein 52B TMEM59 ENSG00000116209 Transmembrane protein59 TMEM67 ENSG00000164953 Transmembrane protein 67 TMEM70ENSG00000175606 Transmembrane protein 70 TMEM87A ENSG00000103978Transmembrane protein 87A TMEM94 ENSG00000177728 Transmembrane protein94 TMEM95 ENSG00000182896 Transmembrane protein 95 TMIGD1ENSG00000182271 Transmembrane and immunoglobulin domain containing 1TMPRSS12 ENSG00000186452 Transmembrane (C-terminal) protease, serine 12TMPRSS5 ENSG00000166682 Transmembrane protease, serine 5 TMUB1ENSG00000164897 Transmembrane and ubiquitin-like domain containing 1TMX2 ENSG00000213593 Thioredoxin-related transmembrane protein 2 TMX3ENSG00000166479 Thioredoxin-related transmembrane protein 3 TNCENSG00000041982 Tenascin C TNFAIP6 ENSG00000123610 Tumor necrosisfactor, alpha-induced protein 6 TNFRSF11A ENSG00000141655 Tumor necrosisfactor receptor superfamily, member 11a, NFKB activator TNFRSF11BENSG00000164761 Tumor necrosis factor receptor superfamily, member 11bTNFRSF12A ENSG00000006327 Tumor necrosis factor receptor superfamily,member 12A TNFRSF14 ENSG00000157873 Tumor necrosis factor receptorsuperfamily, member 14 TNFRSF18 ENSG00000186891 Tumor necrosis factorreceptor superfamily, member 18 TNFRSF1A ENSG00000067182 Tumor necrosisfactor receptor superfamily, member lA TNFRSF1B ENSG00000028137 Tumornecrosis factor receptor superfamily, member 1B TNFRSF25 ENSG00000215788Tumor necrosis factor receptor superfamily, member 25 TNFRSF6BENSG00000243509 Tumor necrosis factor receptor superfamily, member 6b,decoy TNFSF11 ENSG00000120659 Tumor necrosis factor (ligand)superfamily, member 11 TNFSF12 ENSG00000239697 Tumor necrosis factor(ligand) superfamily, member 12 TNFSF12- ENSG00000248871 TNFSF12-TNFSF13readthrough TNFSF13 TNFSF15 ENSG00000181634 Tumor necrosis factor(ligand) superfamily, member 15 TNN ENSG00000120332 Tenascin N TNRENSG00000116147 Tenascin R TNXB ENSG00000168477 Tenascin XB TOMM7ENSG00000196683 Translocase of outer mitochondrial membrane 7 homolog(yeast) TOP1MT ENSG00000184428 Topoisomerase (DNA) I, mitochondrialTOR1A ENSG00000136827 Torsin family 1, member A (torsin A) TOR1BENSG00000136816 Torsin family 1, member B (torsin B) TOR2AENSG00000160404 Torsin family 2, member A TOR3A ENSG00000186283 Torsinfamily 3, member A TPD52 ENSG00000076554 Tumor protein D52 TPOENSG00000115705 Thyroid peroxidase TPP1 ENSG00000166340 Tripeptidylpeptidase I TPSAB1 ENSG00000172236 Tryptase alpha/beta 1 TPSB2ENSG00000197253 Tryptase beta 2 (gene/pseudogene) TPSD1 ENSG00000095917Tryptase delta 1 TPST1 ENSG00000169902 Tyrosylprotein sulfotransferase 1TPST2 ENSG00000128294 Tyrosylprotein sulfotransferase 2 TRABD2AENSG00000186854 TraB domain containing 2A TRABD2B ENSG00000269113 TraBdomain containing 2B TREH ENSG00000118094 Trehalase (brush-bordermembrane glycoprotein) TREM1 ENSG00000124731 Triggering receptorexpressed on myeloid cells 1 TREM2 ENSG00000095970 Triggering receptorexpressed on myeloid cells 2 TRH ENSG00000170893 Thyrotropin-releasinghormone TRIM24 ENSG00000122779 Tripartite motif containing 24 TRIM28ENSG00000130726 Tripartite motif containing 28 TRIO ENSG00000038382 TrioRho guanine nucleotide exchange factor TRNP1 ENSG00000253368TMF1-regulated nuclear protein 1 TSC22D4 ENSG00000166925 T5C22 domainfamily, member 4 TSHB ENSG00000134200 Thyroid stimulating hormone, betaTSHR ENSG00000165409 Thyroid stimulating hormone receptor TSKUENSG00000182704 Tsukushi, small leucine rich proteoglycan TSLPENSG00000145777 Thymic stromal lymphopoietin TSPAN3 ENSG00000140391Tetraspanin 3 TSPAN31 ENSG00000135452 Tetraspanin 31 TSPEARENSG00000175894 Thrombospondin-type laminin G domain and EAR repeatsTTC13 ENSG00000143643 Tetratricopeptide repeat domain 13 TTC19ENSG00000011295 Tetratricopeptide repeat domain 19 TTC9B ENSG00000174521Tetratricopeptide repeat domain 9B TTLL11 ENSG00000175764 Tubulintyrosine ligase-like family member 11 TTR ENSG00000118271 TransthyretinTWSG1 ENSG00000128791 Twisted gastrulation BMP signaling modulator 1TXNDC12 ENSG00000117862 Thioredoxin domain containing 12 (endoplasmicreticulum) TXNDC15 ENSG00000113621 Thioredoxin domain containing 15TXNDC5 ENSG00000239264 Thioredoxin domain containing 5 (endoplasmicreticulum) TXNRD2 ENSG00000184470 Thioredoxin reductase 2 TYRP1ENSG00000107165 Tyrosinase-related protein 1 UBAC2 ENSG00000134882 UBAdomain containing 2 UBALD1 ENSG00000153443 UBA-like domain containing 1UBAP2 ENSG00000137073 Ubiquitin associated protein 2 UBXN8ENSG00000104691 UBX domain protein 8 UCMA ENSG00000165623 Upper zone ofgrowth plate and cartilage matrix associated UCN ENSG00000163794Urocortin UCN2 ENSG00000145040 Urocortin 2 UCN3 ENSG00000178473Urocortin 3 UGGT2 ENSG00000102595 UDP-glucose glycoproteinglucosyltransferase 2 UGT1A10 ENSG00000242515 UDPglucuronosyltransferase 1 family, polypeptide A10 UGT2A1 ENSG00000173610UDP glucuronosyltransferase 2 family, polypeptide A1, complex locusUGT2B11 ENSG00000213759 UDP glucuronosyltransferase 2 family,polypeptide B11 UGT2B28 ENSG00000135226 UDP glucuronosyltransferase 2family, polypeptide B28 UGT2B4 ENSG00000156096 UDPglucuronosyltransferase 2 family, polypeptide B4 UGT2B7 ENSG00000171234UDP glucuronosyltransferase 2 family, polypeptide B7 UGT3A1ENSG00000145626 UDP glycosyltransferase 3 family, polypeptide A1 UGT3A2ENSG00000168671 UDP glycosyltransferase 3 family, polypeptide A2 UGT8ENSG00000174607 UDP glycosyltransferase 8 ULBP3 ENSG00000131019 UL16binding protein 3 UMOD ENSG00000169344 Uromodulin UNC5C ENSG00000182168Unc-5 netrin receptor C UPK3B ENSG00000243566 Uroplakin 3B USP11ENSG00000102226 Ubiquitin specific peptidase 11 USP14 ENSG00000101557Ubiquitin specific peptidase 14 (tRNA-guanine transglycosylase) USP3ENSG00000140455 Ubiquitin specific peptidase 3 UTS2 ENSG00000049247Urotensin 2 UTS2B ENSG00000188958 Urotensin 2B UTY ENSG00000183878Ubiquitously transcribed tetratricopeptide repeat containing, Y- linkedUXS1 ENSG00000115652 UDP-glucuronate decarboxylase 1 VASH1ENSG00000071246 Vasohibin 1 VCAN ENSG00000038427 Versican VEGFAENSG00000112715 Vascular endothelial growth factor A VEGFBENSG00000173511 Vascular endothelial growth factor B VEGFCENSG00000150630 Vascular endothelial growth factor C VGF ENSG00000128564VGF nerve growth factor inducible VIP ENSG00000146469 Vasoactiveintestinal peptide VIPR2 ENSG00000106018 Vasoactive intestinal peptidereceptor 2 VIT ENSG00000205221 Vitrin VKORC1 ENSG00000167397 Vitamin Kepoxide reductase complex, subunit 1 VLDLR ENSG00000147852 Very lowdensity lipoprotein receptor VM01 ENSG00000182853 Vitelline membraneouter layer 1 homolog (chicken) VNN1 ENSG00000112299 Vanin 1 VNN2ENSG00000112303 Vanin 2 VNN3 ENSG00000093134 Vanin 3 VOPP1ENSG00000154978 Vesicular, overexpressed in cancer, prosurvival protein1 VPREB1 ENSG00000169575 Pre-B lymphocyte 1 VPREB3 ENSG00000128218 Pre-Blymphocyte 3 VPS37B ENSG00000139722 Vacuolar protein sorting 37 homologB (S. cerevisiae) VPS51 ENSG00000149823 Vacuolar protein sorting 51homolog (S. cerevisiae) VSIG1 ENSG00000101842 V-set and immunoglobulindomain containing 1 VSIG10 ENSG00000176834 V-set and immunoglobulindomain containing 10 VSTM1 ENSG00000189068 V-set and transmembranedomain containing 1 VSTM2A ENSG00000170419 V-set and transmembranedomain containing 2A VSTM2B ENSG00000187135 V-set and transmembranedomain containing 2B VSTM2L ENSG00000132821 V-set and transmembranedomain containing 2 like VSTM4 ENSG00000165633 V-set and transmembranedomain containing 4 VTN ENSG00000109072 Vitronectin VWA1 ENSG00000179403Von Willebrand factor A domain containing 1 VWA2 ENSG00000165816 VonWillebrand factor A domain containing 2 VWA5B2 ENSG00000145198 VonWillebrand factor A domain containing 5B2 VWA7 ENSG00000204396 VonWillebrand factor A domain containing 7 VWC2 ENSG00000188730 VonWillebrand factor C domain containing 2 VWC2L ENSG00000174453 VonWillebrand factor C domain containing protein 2-like VWCEENSG00000167992 Von Willebrand factor C and EGF domains VWDEENSG00000146530 Von Willebrand factor D and EGF domains VWFENSG00000110799 Von Willebrand factor WDR25 ENSG00000176473 WD repeatdomain 25 WDR81 ENSG00000167716 WD repeat domain 81 WDR90ENSG00000161996 WD repeat domain 90 WFDC1 ENSG00000103175 WAPfour-disulfide core domain 1 WFDC10A ENSG00000180305 WAP four-disulfidecore domain 10A WFDC10B ENSG00000182931 WAP four-disulfide core domain10B WFDC11 ENSG00000180083 WAP four-disulfide core domain 11 WFDC12ENSG00000168703 WAP four-disulfide core domain 12 WFDC13 ENSG00000168634WAP four-disulfide core domain 13 WFDC2 ENSG00000101443 WAPfour-disulfide core domain 2 WFDC3 ENSG00000124116 WAP four-disulfidecore domain 3 WFDC5 ENSG00000175121 WAP four-disulfide core domain 5WFDC6 ENSG00000243543 WAP four-disulfide core domain 6 WFDC8ENSG00000158901 WAP four-disulfide core domain 8 WFIKKN1 ENSG00000127578WAP, follistatin/kazal, immunoglobulin, kunitz and netrin domaincontaining 1 WFIKKN2 ENSG00000173714 WAP, follistatin/kazal,immunoglobulin, kunitz and netrin domain containing 2 WIF1ENSG00000156076 WNT inhibitory factor 1 WISP1 ENSG00000104415 WNT1inducible signaling pathway protein 1 WISP2 ENSG00000064205 WNT1inducible signaling pathway protein 2 WISP3 ENSG00000112761 WNT1inducible signaling pathway protein 3 WNK1 ENSG00000060237 WNK lysinedeficient protein kinase 1 WNT1 ENSG00000125084 Wingless-type MMTVintegration site family, member 1 WNT10B ENSG00000169884 Wingless-typeMMTV integration site family, member 10B WNT11 ENSG00000085741Wingless-type MMTV integration site family, member 11 WNT16ENSG00000002745 Wingless-type MMTV integration site family, member 16WNT2 ENSG00000105989 Wingless-type MMTV integration site family member 2WNT3 ENSG00000108379 Wingless-type MMTV integration site family, member3 WNT3A ENSG00000154342 Wingless-type MMTV integration site family,member 3A WNT5A ENSG00000114251 Wingless-type MMTV integration sitefamily, member 5A WNT5B ENSG00000111186 Wingless-type MMTV integrationsite family, member 5B WNT6 ENSG00000115596 Wingless-type MMTVintegration site family, member 6 WNT7A ENSG00000154764 Wingless-typeMMTV integration site family, member 7A WNT7B ENSG00000188064Wingless-type MMTV integration site family, member 7B WNT8AENSG00000061492 Wingless-type MMTV integration site family, member 8AWNT8B ENSG00000075290 Wingless-type MMTV integration site family, member8B WNT9A ENSG00000143816 Wingless-type MMTV integration site family,member 9A WNT9B ENSG00000158955 Wingless-type MMTV integration sitefamily, member 9B WSB1 ENSG00000109046 WD repeat and SOCS box containing1 WSCD1 ENSG00000179314 WSC domain containing 1 WSCD2 ENSG00000075035WSC domain containing 2 XCL1 ENSG00000143184 Chemokine (C motif) ligand1 XCL2 ENSG00000143185 Chemokine (C motif) ligand 2 XPNPEP2ENSG00000122121 X-prolyl aminopeptidase (aminopeptidase P) 2, membrane-bound XXbac- ENSG00000244255 BPG116M5.17 XXbac- ENSG00000248993BPG181M17.5 XXbac-BPG3213.20 ENSG00000204422 XXYLT1 ENSG00000173950Xyloside xylosyltransferase 1 XYLT1 ENSG00000103489 Xylosyltransferase IXYLT2 ENSG00000015532 Xylosyltransferase II ZFYVE21 ENSG00000100711 Zincfinger, FYVE domain containing 21 ZG16 ENSG00000174992 Zymogen granuleprotein 16 ZG16B ENSG00000162078 Zymogen granule protein 16B ZIC4ENSG00000174963 Zic family member 4 ZNF207 ENSG00000010244 Zinc fingerprotein 207 ZNF26 ENSG00000198393 Zinc finger protein 26 ZNF34ENSG00000196378 Zinc finger protein 34 ZNF419 ENSG00000105136 Zincfinger protein 419 ZNF433 ENSG00000197647 Zinc finger protein 433 ZNF449ENSG00000173275 Zinc finger protein 449 ZNF488 ENSG00000265763 Zincfinger protein 488 ZNF511 ENSG00000198546 Zinc finger protein 511 ZNF570ENSG00000171827 Zinc finger protein 570 ZNF691 ENSG00000164011 Zincfinger protein 691 ZNF98 ENSG00000197360 Zinc finger protein 98 ZPBPENSG00000042813 Zona pellucida binding protein ZPBP2 ENSG00000186075Zona pellucida binding protein 2 ZSCAN29 ENSG00000140265 Zinc finger andSCAN domain containing 29

In some embodiments of the disclosure, T cells are modified to expresstherapeutic proteins, including secreted proteins and secreted humanproteins. In some embodiments of the methods of the disorder,compositions comprising CAR-T cells modified to express or to secrete ahuman protein are used to treat a clotting disorder. Blood clottingoccurs through a multistep process known as the coagulation cascade. Inthe extrinsic pathway, Tissue Factor (also known as factor III orthromboplastin) comes into contact with factor VII to form an activatedVIIa complex. This initiates a coagulation protease cascade, convertingthe inactive Factor X to an active protease Factor Xa, which, withactivated Factor V, produces thrombin (IIa) from Prothrombin (II). Inthe intrinsic pathway, collagen forms a complex withhigh-molecular-weight-kininogen, prekallikrein and Factor XII, leadingto the conversion of Factor XII into Factor XIIa. Factor XIIa convertsFactor XI into Factor XIa, and Factor XIa activates Factor IX to produceFactor IXa, which, together with FVIIIa form the tenase complex, whichactivates Factor X, which helps convert Prothrombin (II) into Thrombin(IIa). Thrombin in turn leads to the conversion of Fibrinogen (I) intoFibrin, which together with Factor XIIIa forms a cross-linked fibrinclot. Many clotting disorders are the result of low levels of secretedproteins in the blood that are involved in the coagulation cascade.Clotting disorders can drastically increase the amount of blood leavingthe body upon injury, or cause bleeding to occur under the skin or invital organs. These disorders are frequently genetic. Exemplary, butnon-limiting diseases caused by deficiencies in clotting factors includeHemophilias, von Willebrand disease and deficiencies in AntithrombinIII, protein C or protein S. Hemophila A and B are X-linked, and arecaused by insufficient levels of clotting factor VIII and factor IX(FIX) respectively. Hemophila C is caused by insufficient factor XI.Factor II, VII, X or XII deficiencies can also cause bleeding disorders.Von Willebrand disease is due to a low level of the von Willebrandclotting factor in the blood. In some cases, deficiencies in bloodproteins that regulate clotting lead can lead to too much clotting.Factor V Leiden is a genetic disorder, where the factor V Leiden proteinoverreacts, causing the blood to clot too often or too much.Deficiencies in Antithrombin III, protein C or protein S, which helpregulate bleeding, can also cause excessive clotting. Currently,clotting disorders such as Hemophilia are treated with bloodtransfusions or infusions of the missing clotting factor (replacementtherapy). However, complications of replacement therapy includedeveloping antibodies to the clotting factor, contracting viralinfections from blood derived products and damage to joints. There thusexists a need for additional therapies.

In some embodiments of the disclosure, T cells are modified to expresstherapeutic proteins, including secreted proteins and secreted humanproteins. In some embodiments of the methods of the disorder,compositions comprising CAR-T cells modified to express or to secrete ahuman protein are used for enzyme replacement therapy. Enzymereplacement therapy typically involves intravenous infusions oftherapeutically effective amounts of compositions comprising enzymesthat balance underlying enzyme deficiencies that cause the symptoms ofthe disease. The missing enzyme activity is thus supplied exogenously inthis manner. Exemplary diseases that can be treated by modified T cellsof the disclosure include, but are not limited to, lysosomal storagediseases Gaucher's disease (glucocerebrosidase enzyme), Fabry disease,mucopolysaccharidosis I (MPS I), mucopolysaccharidosis I (MPS II, orHunter syndrome, caused by iduronate-2-sulfatase deficiency),mucopolysaccharidosis VI (MPS VI, caused by arylsulfatase B deficiency)and Pompe disease (or glycogen stoarage disease type II, caused by adeficiency in acid alpha-glucosidase). Additional diseases treatablewith enzyme replacement therapy include but are not limited to Adenosinedeaminase (ADA) deficiency, Hyperammonemia due to the deficiency of thehepatic enzyme N-acetylglutamate synthetase (NAGS), Hypophosphatasia,Lysosomal acid lipase deficiency, Morquio Syndrome A, Wolman LALLysosomal Acid Lipase deficiency, A1AT (Alpha1-Antitrypsin) deficiencyand Urea cycle disorder. Enzymes supplied to patients during enzymereplacement therapy include, but are not limited to Alpha1-Antitrypsin,β-Glucocerebrosidase, Adenosine Deaminase, Alpha-Galactosidase A,α-L-Iduronidase, Iduronate-2-Sulfatase, N-Acetylgalactosamine-6Sulfatase, -Acetylgalactosamine-4 Sulfatase and Lysosomal Acid Lipase.

In some embodiments of the disclosure, T cells are modified to expresstherapeutic proteins, including secreted proteins and secreted humanproteins. In some embodiments of the methods of the disorder,compositions comprising CAR-T cells modified to express or to secrete ahuman protein are used to produce human antibodies. In some embodiments,the disease to be treated by modified T cells expressing secretedproteins is a disease that can be treated through the intravenousinfusion or injection of an antibody or an antibody fragment. Antibodybased therapies are used in the treatment of many types of diseases inaddition to cancer, including immune-based diseases such as arthritisand asthma, and infections, as well as other diseases. Exemplary, butnon-limiting list of diseases that can be treated with the modified Tcells of the disclosure include platelet aggregation, Clostridiumdifficile infection, Rheumatoid arthritis, Crohn's Disease, PlaquePsoriasis, Psoriatic Arthritis, Ankylosing Spondylitis, JuvenileIdiopathic Arthritis, Alzheimer's disease, sepsis, Multiple Sclerosis,hypercholesterolemia, systemic lupus erythematosus, prevention of organtransplant rejections, viral infections, asthma, severe allergicdisorders, retinopathy, osteoporosis, inflammatory bowel diseases,inflammatory diseases, influenza A, paroxysmal nocturnal hemoglobinuria,sepsis caused by Gram-negative bacteria, psoriasis, invasive Candidainfection, ulcerative colitis, hypocholesterolemia, respiratorysyncytial virus infection, focal segmental glomerulosclerosis, graftversus host disease, ankylosing spondylitis, HIV infection, ulcerativecolitis, autoimmune diseases, chronic asthma, reduction of scarringafter glaucoma surgery, hypercholesterolemia, white blood cell diseases,systemic scleroderma, respiratory syncytial virus (prevention), lupuserythematosus, diabetes mellitus type 1, inflammation, Pseudomonasaeruginosa infection, macular degeneration, anthrax, cytomegalovirusinfection, inflammations of the airways, skin and gastrointestinaltract, systemic lupus erythematosus, rheumatic diseases, uveitis,cytomegalovirus infection, dermatomyositis, polymyositis, fibrosis,choroidal and retinal neovascularization, muscular dystrophy,Staphylococcus aureus infection, lupus nephritis, follicular lymphoma,chronic hepatitis B and ulcerative colitis.

Infusion of Modified Cells as Adoptive Cell Therapy

In certain embodiments of the disclosure, modified cells of thedisclosure are delivered to a patient via injection or intravenousinfusion. In certain embodiments, a therapeutically effective dose of acomposition of the disclosure or of compositions comprising modifiedcells of the disclosure comprises between 2×10⁵ and 5×10⁸ cells per kgof body weight of the patient per administration, or any range, value orfraction thereof.

In certain embodiments of the disclosure, modified cells of thedisclosure are delivered to a patient via injection or intravenousinfusion. In certain embodiments, a therapeutically effective dose of acomposition of the disclosure or of compositions comprising modifiedcells of the disclosure comprises between 0.2×10⁶ to 20×10⁶ cells per kgof body weight of the patient per administration. In certainembodiments, a therapeutically effective dose of a composition of thedisclosure or of compositions comprising modified cells of thedisclosure comprises 0.2×10⁶ cells per kg of body weight of the patientper administration, 2×10⁶ cells per kg of body weight of the patient peradministration, 20×10⁶ cells per kg of body weight of the patient peradministration, or any cells per kg of body weight of the patient peradministration in between.

In certain embodiments of the disclosure, modified cells of thedisclosure are delivered to a patient via injection or intravenousinfusion. In certain embodiments, a therapeutically effective dose of acomposition of the disclosure or of compositions comprising modifiedcells of the disclosure comprises 1×10⁶ cells or about 1×10⁶ cells perkg of body weight of the patient per administration.

In certain embodiments of the disclosure, modified cells of thedisclosure are delivered to a patient via injection or intravenousinfusion. In certain embodiments, a therapeutically effective dose of acomposition of the disclosure or of compositions comprising modifiedcells of the disclosure comprises 3×10⁶ cells or about 3×10⁶ cells perkg of body weight of the patient per administration.

In certain embodiments of the disclosure, modified cells of thedisclosure are delivered to a patient via injection or intravenousinfusion. In certain embodiments, a therapeutically effective dose of acomposition of the disclosure or of compositions comprising modifiedcells of the disclosure comprises between 0.7×10⁶ to 6.7×10⁶ cells perkg of body weight of the patient per administration. In certainembodiments, a therapeutically effective dose of a composition of thedisclosure or of compositions comprising modified cells of thedisclosure comprises 0.7×10⁶ cells per kg of body weight of the patientper administration, 6.7×10⁶ cells per kg of body weight of the patientper administration or any cells per kg of body weight of the patient peradministration in between.

In certain embodiments of the disclosure, modified cells of thedisclosure are delivered to a patient via injection or intravenousinfusion. In certain embodiments, a therapeutically effective dose of acomposition of the disclosure or of compositions comprising modifiedcells of the disclosure comprises between 0.7×10⁶ to 16×10⁶ cells per kgof body weight of the patient per administration. In certainembodiments, a therapeutically effective dose of a composition of thedisclosure or of compositions comprising modified cells of thedisclosure comprises 0.7×10⁶ cells per kg of body weight of the patientper administration, 2×10⁶ cells per kg of body weight of the patient peradministration, 6×10⁶ cells per kg of body weight of the patient peradministration, 10.7×10⁶ cells per kg of body weight of the patient peradministration, 16×10⁶ cells per kg of body weight of the patient peradministration or any cells per kg of body weight of the patient peradministration in between.

In certain embodiments of the disclosure, modified cells of thedisclosure are delivered to a patient via injection or intravenousinfusion. In certain embodiments, a therapeutically effective dose of acomposition of the disclosure or of compositions comprising modifiedcells of the disclosure comprises 1.2×10⁶ to 7.1×10⁶ cells per kg ofbody weight of the patient per administration. In certain embodiments, atherapeutically effective dose of a composition of the disclosure or ofcompositions comprising modified cells of the disclosure comprises1.2×10⁶ cells per kg of body weight of the patient per administration,7.1×10⁶ cells per kg of body weight of the patient per administration orany number of cells per kg of body weight of the patient peradministration. In certain embodiments, a therapeutically effective doseof a composition of the disclosure or of compositions comprisingmodified cells of the disclosure comprises between 2×10⁶ to 3×10⁶ cellsper kg of body weight of the patient per administration.

In certain embodiments of the disclosure, modified cells of thedisclosure are delivered to a patient via injection or intravenousinfusion. In certain embodiments, a therapeutically effective dose of acomposition of the disclosure or of compositions comprising modifiedcells of the disclosure comprises 1106×10⁶ to 2106×10⁶ cells per kg ofbody weight of the patient per administration. In certain embodiments, atherapeutically effective dose of a composition of the disclosure or ofcompositions comprising modified cells of the disclosure comprises1106×10⁶ cells per kg of body weight of the patient per administration,2106×10⁶ cells per kg of body weight of the patient per administrationor any number of cells per kg of body weight of the patient peradministration in between. In certain embodiments of the disclosure,modified cells of the disclosure are delivered to a patient viainjection or intravenous infusion. In certain embodiments, atherapeutically effective dose of a composition of the disclosure or ofcompositions comprising modified cells of the disclosure comprises0.7×10⁶ to 1.3×10⁶ cells per kg of body weight of the patient peradministration. In certain embodiments, a therapeutically effective doseof a composition of the disclosure or of compositions comprisingmodified cells of the disclosure comprises 0.7×10⁶ cells per kg of bodyweight of the patient per administration, 1.3×10⁶ cells per kg of bodyweight of the patient per administration or any number of cells per kgof body weight of the patient per administration in between.

In certain embodiments of the disclosure, modified cells of thedisclosure are delivered to a patient via injection or intravenousinfusion. In certain embodiments, a therapeutically effective dose of acomposition of the disclosure or of compositions comprising modifiedcells of the disclosure comprises a single or multiple doses. In certainembodiments, a therapeutically effective dose of a composition of thedisclosure or of compositions comprising modified cells of thedisclosure comprises a split dose. In certain embodiments, atherapeutically effective dose of a composition of the disclosure or ofcompositions comprising modified cells of the disclosure comprises aninitial dose and a maintenance dose.

In certain embodiments of the disclosure, the modified cells are T cellsand the T cells may be sorted according to T cell markers prior toeither in vitro expansion or formulation with a pharmaceuticallyacceptable carrier. In some embodiments, modified T cells may be sortedon using CD8+ and/or CD4+ markers.

Nucleic Acid Molecules

Nucleic acid molecules of the disclosure encoding protein scaffolds canbe in the form of RNA, such as mRNA, hnRNA, tRNA or any other form, orin the form of DNA, including, but not limited to, cDNA and genomic DNAobtained by cloning or produced synthetically, or any combinationsthereof. The DNA can be triple-stranded, double-stranded orsingle-stranded, or any combination thereof. Any portion of at least onestrand of the DNA or RNA can be the coding strand, also known as thesense strand, or it can be the non-coding strand, also referred to asthe anti-sense strand.

Isolated nucleic acid molecules of the disclosure can include nucleicacid molecules comprising an open reading frame (ORF), optionally, withone or more introns, e.g., but not limited to, at least one specifiedportion of at least one protein scaffold; nucleic acid moleculescomprising the coding sequence for a protein scaffold or loop regionthat binds to the target protein; and nucleic acid molecules whichcomprise a nucleotide sequence substantially different from thosedescribed above but which, due to the degeneracy of the genetic code,still encode the protein scaffold, Centyrin, CAR, CARTyrin, transposon,and/or transposase as described herein and/or as known in the art. Ofcourse, the genetic code is well known in the art. Thus, it would beroutine for one skilled in the art to generate such degenerate nucleicacid variants that code for specific protein scaffolds of the presentinvention. See, e.g., Ausubel, et al., supra, and such nucleic acidvariants are included in the present invention.

As indicated herein, nucleic acid molecules of the disclosure whichcomprise a nucleic acid encoding a protein scaffold, Centyrin, CAR,CARTyrin, transposon, and/or transposase can include, but are notlimited to, those encoding the amino acid sequence of a proteinscaffold, Centyrin, CAR, CARTyrin, transposon, and/or transposasefragment, by itself; the coding sequence for the entire proteinscaffold, Centyrin, CAR, CARTyrin, transposon, and/or transposase or aportion thereof; the coding sequence for a protein scaffold, Centyrin,CAR, CARTyrin, transposon, and/or transposase, fragment or portion, aswell as additional sequences, such as the coding sequence of at leastone signal leader or fusion peptide, with or without the aforementionedadditional coding sequences, such as at least one intron, together withadditional, non-coding sequences, including but not limited to,non-coding 5′ and 3′ sequences, such as the transcribed, non-translatedsequences that play a role in transcription, mRNA processing, includingsplicing and polyadenylation signals (for example, ribosome binding andstability of mRNA); an additional coding sequence that codes foradditional amino acids, such as those that provide additionalfunctionalities. Thus, the sequence encoding a protein scaffold,Centyrin, CAR, CARTyrin, transposon, and/or transposase can be fused toa marker sequence, such as a sequence encoding a peptide thatfacilitates purification of the fused protein scaffold, Centyrin, CAR,CARTyrin, transposon, and/or transposase comprising a protein scaffoldfragment or portion.

Construction of Nucleic Acids

The isolated nucleic acids of the disclosure can be made using (a)recombinant methods, (b) synthetic techniques, (c) purificationtechniques, and/or (d) combinations thereof, as well-known in the art.

The nucleic acids can conveniently comprise sequences in addition to apolynucleotide of the present invention. For example, a multi-cloningsite comprising one or more endonuclease restriction sites can beinserted into the nucleic acid to aid in isolation of thepolynucleotide. Also, translatable sequences can be inserted to aid inthe isolation of the translated polynucleotide of the disclosure. Forexample, a hexa-histidine marker sequence provides a convenient means topurify the proteins of the disclosure. The nucleic acid of thedisclosure, excluding the coding sequence, is optionally a vector,adapter, or linker for cloning and/or expression of a polynucleotide ofthe disclosure.

Additional sequences can be added to such cloning and/or expressionsequences to optimize their function in cloning and/or expression, toaid in isolation of the polynucleotide, or to improve the introductionof the polynucleotide into a cell. Use of cloning vectors, expressionvectors, adapters, and linkers is well known in the art. (See, e.g.,Ausubel, supra; or Sambrook, supra).

Recombinant Methods for Constructing Nucleic Acids

The isolated nucleic acid compositions of this disclosure, such as RNA,cDNA, genomic DNA, or any combination thereof, can be obtained frombiological sources using any number of cloning methodologies known tothose of skill in the art. In some embodiments, oligonucleotide probesthat selectively hybridize, under stringent conditions, to thepolynucleotides of the present invention are used to identify thedesired sequence in a cDNA or genomic DNA library. The isolation of RNA,and construction of cDNA and genomic libraries are well known to thoseof ordinary skill in the art. (See, e.g., Ausubel, supra; or Sambrook,supra).

Vectors and Host Cells

The disclosure also relates to vectors that include isolated nucleicacid molecules of the disclosure, host cells that are geneticallyengineered with the recombinant vectors, and the production of at leastone protein scaffold by recombinant techniques, as is well known in theart. See, e.g., Sambrook, et al., supra; Ausubel, et al., supra, eachentirely incorporated herein by reference.

For example, the PB-EF1a vector may be used. The vector comprises thefollowing nucleotide sequence:

(SEQ ID NO: 35)tgtacatagattaaccctagaaagataatcatattgtgacgtacgttaaagataatcatgcgtaaaattgacgcatgtgttttatcggtctgtatatcgaggtttatttattaatttgaatagatattaagttttattatatttacacttacatactaataataaattcaacaaacaatttatttatgtttatttatttattaaaaaaaaacaaaaactcaaaatttcttctataaagtaacaaaacttttatcgaatacctgcagcccgggggatgcagagggacagcccccccccaaagcccccagggatgtaattacgtccctcccccgctagggggcagcagcgagccgcccggggctccgctccggtccggcgctccccccgcatccccgagccggcagcgtgcggggacagcccgggcacggggaaggtggcacgggatcgctttcctctgaacgcttctcgctgctctttgagcctgcagacacctggggggatacggggaaaagttgactgtgcctttcgatcgaaccatggacagttagctttgcaaagatggataaagttttaaacagagaggaatctttgcagctaatggaccttctaggtcttgaaaggagtgggaattggctccggtgcccgtcagtgggcagagcgcacatcgcccacagtccccgagaagttggggggaggggtcggcaattgaaccggtgcctagagaaggtggcgcggggtaaactgggaaagtgatgtcgtgtactggctccgcctttttcccgagggtgggggagaaccgtatataagtgcagtagtcgccgtgaacgttctttttcgcaacgggtttgccgccagaacacaggtaagtgccgtgtgtggttcccgcgggcctggcctctttacgggttatggcccttgcgtgccttgaattacttccacctggctgcagtacgtgattcttgatcccgagcttcgggttggaagtgggtgggagagttcgaggccttgcgcttaaggagccccttcgcctcgtgcttgagttgaggcctggcctgggcgctggggccgccgcgtgcgaatctggtggcaccttcgcgcctgtctcgctgctttcgataagtctctagccatttaaaatttttgatgacctgctgcgacgctttttttctggcaagatagtcttgtaaatgcgggccaagatctgcacactggtatttcggtttttggggccgcgggcggcgacggggcccgtgcgtcccagcgcacatgttcggcgaggcggggcctgcgagcgcggccaccgagaatcggacgggggtagtctcaagctggccggcctgctctggtgcctggcctcgcgccgccgtgtatcgccccgccctgggcggcaaggctggcccggtcggcaccagttgcgtgagcggaaagatggccgcttcccggccctgctgcagggagctcaaaatggaggacgcggcgctcgggagagcgggcgggtgagtcacccacacaaaggaaaagggcctttccgtcctcagccgtcgcttcatgtgactccacggagtaccgggcgccgtccaggcacctcgattagttctcgagcttttggagtacgtcgtctttaggttggggggaggggttttatgcgatggagtttccccacactgagtgggtggagactgaagttaggccagcttggcacttgatgtaattctccttggaatttgccctttttgagtttggatcttggttcattctcaagcctcagacagtggttcaaagtttttttcttccatttcaggtgtcgtgagaattctaatacgactcactatagggtgtgctgtctcatcattttggcaaagattggccaccaagcttgtcctgcaggagggtcgacgcctctagacgggcggccgctccggatccacgggtaccgatcacatatgcctttaattaaacactagttctatagtgtcacctaaattccctttagtgagggttaatggccgtaggccgccagaattgggtccagacatgataagatacattgatgagtttggacaaaccacaactagaatgcagtgaaaaaaatgctttatttgtgaaatttgtgatgctattgctttatttgtaaccattataagctgcaataaacaagttaacaacaacaattgcattcattttatgtttcaggttcagggggaggtgtgggaggttttttcggactctaggacctgcgcatgcgcttggcgtaatcatggtcatagctgtttcctgttttccccgtatccccccaggtgtctgcaggctcaaagagcagcgagaagcgttcagaggaaagcgatcccgtgccaccttccccgtgcccgggctgtccccgcacgctgccggctcggggatgcggggggagcgccggaccggagcggagccccgggcggctcgctgctgccccctagcgggggagggacgtaattacatccctgggggctttgggggggggctgtccctctcaccgcggtggagctccagcttttgttcgaattggggccccccctcgagggtatcgatgatatctataacaagaaaatatatatataataagttatcacgtaagtagaacatgaaataacaatataattatcgtatgagttaaatcttaaaagtcacgtaaaagataatcatgcgtcattttgactcacgcggtcgttatagttcaaaatcagtgacacttaccgcattgacaagcacgcctcacgggagctccaagcggcgactgagatgtcctaaatgcacagcgacggattcgcgctatttagaaagagagagcaatatttcaagaatgcatgcgtcaattttacgcagactatctttctagggttaatctagctagccttaagggcgcctattgcgttgcgctcactgcccgctttccagtcgggaaacctgtcgtgccagctgcattaatgaatcggccaacgcgcggggagaggcggtttgcgtattgggcgctcttccgcttcctcgctcactgactcgctgcgctcggtcgttcggctgcggcgagcggtatcagctcactcaaaggcggtaatacggttatccacagaatcaggggataacgcaggaaagaacatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgttcttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctggccttttgctcacatgagattatcaaaaaggatcttcacctagatccttttaaattaaaaatgaagttttaaatcaatctaaagtatatatgagtaaacttggtctgacagtcagaagaactcgtcaagaaggcgatagaaggcgatgcgctgcgaatcgggagcggcgataccgtaaagcacgaggaagcggtcagcccattcgccgccaagctcttcagcaatatcacgggtagccaacgctatgtcctgatagcggtccgccacacccagccggccacagtcgatgaatccagaaaagcggccattttccaccatgatattcggcaagcaggcatcgccatgggtcacgacgagatcctcgccgtcgggcatgctcgccttgagcctggcgaacagttcggctggcgcgagcccctgatgctcttcgtccagatcatcctgatcgacaagaccggcttccatccgagtacgtgctcgctcgatgcgatgtttcgcttggtggtcgaatgggcaggtagccggatcaagcgtatgcagccgccgcattgcatcagccatgatggatactttctcggcaggagcaaggtgagatgacaggagatcctgccccggcacttcgcccaatagcagccagtcccttcccgcttcagtgacaacgtcgagcacagctgcgcaaggaacgcccgtcgtggccagccacgatagccgcgctgcctcgtcttgcagttcattcagggcaccggacaggtcggtcttgacaaaaagaaccgggcgcccctgcgctgacagccggaacacggcggcatcagagcagccgattgtctgttgtgcccagtcatagccgaatagcctctccacccaagcggccggagaacctgcgtgcaatccatcttgttcaatcataatattattgaagcatttatcagggttcgtctcgtcccggtctcctcccaatgcatgtcaatattggccattagccatattattcattggttatatagcataaatcaatattggctattggccattgcatacgttgtatctatatcataata.

The polynucleotides can optionally be joined to a vector containing aselectable marker for propagation in a host. Generally, a plasmid vectoris introduced in a precipitate, such as a calcium phosphate precipitate,or in a complex with a charged lipid. If the vector is a virus, it canbe packaged in vitro using an appropriate packaging cell line and thentransduced into host cells.

The DNA insert should be operatively linked to an appropriate promoter.The expression constructs will further contain sites for transcriptioninitiation, termination and, in the transcribed region, a ribosomebinding site for translation. The coding portion of the maturetranscripts expressed by the constructs will preferably include atranslation initiating at the beginning and a termination codon (e.g.,UAA, UGA or UAG) appropriately positioned at the end of the mRNA to betranslated, with UAA and UAG preferred for mammalian or eukaryotic cellexpression.

Expression vectors will preferably but optionally include at least oneselectable marker. Such markers include, e.g., but are not limited to,ampicillin, zeocin (Sh bla gene), puromycin (pac gene), hygromycin B(hygB gene), G418/Geneticin (neo gene), mycophenolic acid, or glutaminesynthetase (GS, U.S. Pat. Nos. 5,122,464; 5,770,359; 5,827,739),blasticidin (bsd gene), resistance genes for eukaryotic cell culture aswell as ampicillin, zeocin (Sh bla gene), puromycin (pac gene),hygromycin B (hygB gene), G418/Geneticin (neo gene), kanamycin,spectinomycin, streptomycin, carbenicillin, bleomycin, erythromycin,polymyxin B, or tetracycline resistance genes for culturing in E. coliand other bacteria or prokaryotics (the above patents are entirelyincorporated hereby by reference). Appropriate culture mediums andconditions for the above-described host cells are known in the art.Suitable vectors will be readily apparent to the skilled artisan.Introduction of a vector construct into a host cell can be effected bycalcium phosphate transfection, DEAE-dextran mediated transfection,cationic lipid-mediated transfection, electroporation, transduction,infection or other known methods. Such methods are described in the art,such as Sambrook, supra, Chapters 1-4 and 16-18; Ausubel, supra,Chapters 1, 9, 13, 15, 16.

Expression vectors will preferably but optionally include at least oneselectable cell surface marker for isolation of cells modified by thecompositions and methods of the disclosure. Selectable cell surfacemarkers of the disclosure comprise surface proteins, glycoproteins, orgroup of proteins that distinguish a cell or subset of cells fromanother defined subset of cells. Preferably the selectable cell surfacemarker distinguishes those cells modified by a composition or method ofthe disclosure from those cells that are not modified by a compositionor method of the disclosure. Such cell surface markers include, e.g.,but are not limited to, “cluster of designation” or “classificationdeterminant” proteins (often abbreviated as “CD”) such as a truncated orfull length form of CD19, CD271, CD34, CD22, CD20, CD33, CD52, or anycombination thereof. Cell surface markers further include the suicidegene marker RQR8 (Philip B et al. Blood. 2014 Aug. 21; 124(8):1277-87).

Expression vectors will preferably but optionally include at least oneselectable drug resistance marker for isolation of cells modified by thecompositions and methods of the disclosure. Selectable drug resistancemarkers of the disclosure may comprise wild-type or mutant Neo, DHFR,TYMS, FRANCF, RAD51C, GCS, MDR1, ALDH1, NKX2.2, or any combinationthereof.

At least one protein scaffold of the disclosure can be expressed in amodified form, such as a fusion protein, and can include not onlysecretion signals, but also additional heterologous functional regions.For instance, a region of additional amino acids, particularly chargedamino acids, can be added to the N-terminus of a protein scaffold toimprove stability and persistence in the host cell, during purification,or during subsequent handling and storage. Also, peptide moieties can beadded to a protein scaffold of the disclosure to facilitatepurification. Such regions can be removed prior to final preparation ofa protein scaffold or at least one fragment thereof. Such methods aredescribed in many standard laboratory manuals, such as Sambrook, supra,Chapters 17.29-17.42 and 18.1-18.74; Ausubel, supra, Chapters 16, 17 and18.

Those of ordinary skill in the art are knowledgeable in the numerousexpression systems available for expression of a nucleic acid encoding aprotein of the disclosure. Alternatively, nucleic acids of thedisclosure can be expressed in a host cell by turning on (bymanipulation) in a host cell that contains endogenous DNA encoding aprotein scaffold of the disclosure. Such methods are well known in theart, e.g., as described in U.S. Pat. Nos. 5,580,734, 5,641,670,5,733,746, and 5,733,761, entirely incorporated herein by reference.

Illustrative of cell cultures useful for the production of the proteinscaffolds, specified portions or variants thereof, are bacterial, yeast,and mammalian cells as known in the art. Mammalian cell systems oftenwill be in the form of monolayers of cells although mammalian cellsuspensions or bioreactors can also be used. A number of suitable hostcell lines capable of expressing intact glycosylated proteins have beendeveloped in the art, and include the COS-1 (e.g., ATCC CRL 1650), COS-7(e.g., ATCC CRL-1651), HEK293, BHK21 (e.g., ATCC CRL-10), CHO (e.g.,ATCC CRL 1610) and BSC-1 (e.g., ATCC CRL-26) cell lines, Cos-7 cells,CHO cells, hep G2 cells, P3X63Ag8.653, SP2/0-Ag14, 293 cells, HeLa cellsand the like, which are readily available from, for example, AmericanType Culture Collection, Manassas, Va. (www.atcc.org). Preferred hostcells include cells of lymphoid origin, such as myeloma and lymphomacells. Particularly preferred host cells are P3X63Ag8.653 cells (ATCCAccession Number CRL-1580) and SP2/0-Ag14 cells (ATCC Accession NumberCRL-1851). In a particularly preferred embodiment, the recombinant cellis a P3X63Ab8.653 or an SP2/0-Ag14 cell.

Expression vectors for these cells can include one or more of thefollowing expression control sequences, such as, but not limited to, anorigin of replication; a promoter (e.g., late or early SV40 promoters,the CMV promoter (U.S. Pat. Nos. 5,168,062; 5,385,839), an HSV tkpromoter, a pgk (phosphoglycerate kinase) promoter, an EF-1 alphapromoter (U.S. Pat. No. 5,266,491), at least one human promoter; anenhancer, and/or processing information sites, such as ribosome bindingsites, RNA splice sites, polyadenylation sites (e.g., an SV40 large T Agpoly A addition site), and transcriptional terminator sequences. See,e.g., Ausubel et al., supra; Sambrook, et al., supra. Other cells usefulfor production of nucleic acids or proteins of the present invention areknown and/or available, for instance, from the American Type CultureCollection Catalogue of Cell Lines and Hybridomas (www.atcc.org) orother known or commercial sources.

When eukaryotic host cells are employed, polyadenlyation ortranscription terminator sequences are typically incorporated into thevector. An example of a terminator sequence is the polyadenlyationsequence from the bovine growth hormone gene. Sequences for accuratesplicing of the transcript can also be included. An example of asplicing sequence is the VP1 intron from SV40 (Sprague, et al., J.Virol. 45:773-781 (1983)). Additionally, gene sequences to controlreplication in the host cell can be incorporated into the vector, asknown in the art.

Purification of a Protein Scaffold

A protein scaffold can be recovered and purified from recombinant cellcultures by well-known methods including, but not limited to, protein Apurification, ammonium sulfate or ethanol precipitation, acidextraction, anion or cation exchange chromatography, phosphocellulosechromatography, hydrophobic interaction chromatography, affinitychromatography, hydroxylapatite chromatography and lectinchromatography. High performance liquid chromatography (“HPLC”) can alsobe employed for purification. See, e.g., Colligan, Current Protocols inImmunology, or Current Protocols in Protein Science, John Wiley & Sons,NY, N.Y., (1997-2001), e.g., Chapters 1, 4, 6, 8, 9, 10, each entirelyincorporated herein by reference.

Protein scaffolds of the disclosure include naturally purified products,products of chemical synthetic procedures, and products produced byrecombinant techniques from a prokaryotic or eukaryotic host, including,for example, E. coli, yeast, higher plant, insect and mammalian cells.Depending upon the host employed in a recombinant production procedure,the protein scaffold of the disclosure can be glycosylated or can benon-glycosylated. Such methods are described in many standard laboratorymanuals, such as Sambrook, supra, Sections 17.37-17.42; Ausubel, supra,Chapters 10, 12, 13, 16, 18 and 20, Colligan, Protein Science, supra,Chapters 12-14, all entirely incorporated herein by reference.

Variants

The amino acids that make up protein scaffolds of the disclosure areoften abbreviated. The amino acid designations can be indicated bydesignating the amino acid by its single letter code, its three lettercode, name, or three nucleotide codon(s) as is well understood in theart (see Alberts, B., et al., Molecular Biology of The Cell, Third Ed.,Garland Publishing, Inc., New York, 1994). A protein scaffold of thedisclosure can include one or more amino acid substitutions, deletionsor additions, either from natural mutations or human manipulation, asspecified herein. Amino acids in a protein scaffold of the disclosurethat are essential for function can be identified by methods known inthe art, such as site-directed mutagenesis or alanine-scanningmutagenesis (e.g., Ausubel, supra, Chapters 8, 15; Cunningham and Wells,Science 244:1081-1085 (1989)). The latter procedure introduces singlealanine mutations at every residue in the molecule. The resulting mutantmolecules are then tested for biological activity, such as, but notlimited to, at least one neutralizing activity. Sites that are criticalfor protein scaffold binding can also be identified by structuralanalysis, such as crystallization, nuclear magnetic resonance orphotoaffinity labeling (Smith, et al., J. Mol. Biol. 224:899-904 (1992)and de Vos, et al., Science 255:306-312 (1992)).

As used throughout the disclosure, the term “substantiallycomplementary” refers to a first sequence that is at least 60%, 65%,70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to thecomplement of a second sequence over a region of 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55,60, 65, 70, 75, 80, 85, 90, 95, 100, 180, 270, 360, 450, 540, or morenucleotides or amino acids, or that the two sequences hybridize understringent hybridization conditions.

As used throughout the disclosure, the term “substantially identical”refers to a first and second sequence are at least 60%, 65%, 70%, 75%,80%, 85%, 90%, 95%, 97%, 98% or 99% identical over a region of 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40,45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 180, 270, 360, 450, 540or more nucleotides or amino acids, or with respect to nucleic acids, ifthe first sequence is substantially complementary to the complement ofthe second sequence.

As used throughout the disclosure, the term “variant” when used todescribe a nucleic acid, refers to (i) a portion or fragment of areferenced nucleotide sequence; (ii) the complement of a referencednucleotide sequence or portion thereof; (iii) a nucleic acid that issubstantially identical to a referenced nucleic acid or the complementthereof; or (iv) a nucleic acid that hybridizes under stringentconditions to the referenced nucleic acid, complement thereof, or asequences substantially identical thereto.

As used throughout the disclosure, the term “vector” refers to a nucleicacid sequence containing an origin of replication. A vector can be aviral vector, a bacteriophage, a bacterial artificial chromosome or ayeast artificial chromosome. A vector can be a DNA or RNA vector. Avector can be a self-replicating extrachromosomal vector, andpreferably, is a DNA plasmid.

As used throughout the disclosure, the term “variant” when used todescribe a peptide or polypeptide, refers to a peptide or polypeptidethat differs in amino acid sequence by the insertion, deletion, orconservative substitution of amino acids, but retain at least onebiological activity. Variant can also mean a protein with an amino acidsequence that is substantially identical to a referenced protein with anamino acid sequence that retains at least one biological activity.

A conservative substitution of an amino acid, i.e., replacing an aminoacid with a different amino acid of similar properties (e.g.,hydrophilicity, degree and distribution of charged regions) isrecognized in the art as typically involving a minor change. These minorchanges can be identified, in part, by considering the hydropathic indexof amino acids, as understood in the art (Kyte et al., J. Mol. Biol.157: 105-132 (1982)). The hydropathic index of an amino acid is based ona consideration of its hydrophobicity and charge. Amino acids of similarhydropathic indexes can be substituted and still retain proteinfunction. In one aspect, amino acids having hydropathic indexes of ±2are substituted. The hydrophilicity of amino acids can also be used toreveal substitutions that would result in proteins retaining biologicalfunction. A consideration of the hydrophilicity of amino acids in thecontext of a peptide permits calculation of the greatest local averagehydrophilicity of that peptide, a useful measure that has been reportedto correlate well with antigenicity and immunogenicity. U.S. Pat. No.4,554,101, incorporated fully herein by reference.

Substitution of amino acids having similar hydrophilicity values canresult in peptides retaining biological activity, for exampleimmunogenicity. Substitutions can be performed with amino acids havinghydrophilicity values within ±2 of each other. Both the hydrophobicityindex and the hydrophilicity value of amino acids are influenced by theparticular side chain of that amino acid. Consistent with thatobservation, amino acid substitutions that are compatible withbiological function are understood to depend on the relative similarityof the amino acids, and particularly the side chains of those aminoacids, as revealed by the hydrophobicity, hydrophilicity, charge, size,and other properties.

As used herein, “conservative” amino acid substitutions may be definedas set out in Tables A, B, or C below. In some embodiments, fusionpolypeptides and/or nucleic acids encoding such fusion polypeptidesinclude conservative substitutions have been introduced by modificationof polynucleotides encoding polypeptides of the invention. Amino acidscan be classified according to physical properties and contribution tosecondary and tertiary protein structure. A conservative substitution isa substitution of one amino acid for another amino acid that has similarproperties. Exemplary conservative substitutions are set out in Table A.

TABLE A Conservative Substitutions I Side chain characteristics AminoAcid Aliphatic Non-polar G A P I L V F Polar - uncharged C S T M N QPolar-charged D E K R Aromatic H F W Y Other N Q D E

Alternately, conservative amino acids can be grouped as described inLehninger, (Biochemistry, Second Edition; Worth Publishers, Inc. NY,N.Y. (1975), pp. 71-77) as set forth in Table B.

TABLE B Conservative Substitutions II Side Chain Characteristic AminoAcid Non-polar (hydrophobic) Aliphatic: A L I V P Aromatic: F W YSulfur-containing: M Borderline: G Y Uncharged-polar Hydroxyl: S T YAmides: N Q Sulfhydryl: C Borderline: G Y Positively Charged (Basic): KR H Negatively Charged (Acidic): D E

Alternately, exemplary conservative substitutions are set out in TableC.

TABLE C Conservative Substitutions III Original Residue ExemplarySubstitution Ala (A) Val Leu Ile Met Arg (R) Lys His Asn (N) Gln Asp (D)Glu Cys (C) Ser Thr Gln (Q) Asn Glu (E) Asp Gly (G) Ala Val Leu Pro His(H) Lys Arg Ile (I) Leu Val Met Ala Phe Leu (L) Ile Val Met Ala Phe Lys(K) Arg His Met (M) Leu Ile Val Ala Phe (F) Trp Tyr Ile Pro (P) Gly AlaVal Leu Ile Ser (S) Thr Thr (T) Ser Trp (W) Tyr Phe Ile Tyr (Y) Trp PheThr Ser Val (V) Ile Leu Met Ala

It should be understood that the polypeptides of the disclosure areintended to include polypeptides bearing one or more insertions,deletions, or substitutions, or any combination thereof, of amino acidresidues as well as modifications other than insertions, deletions, orsubstitutions of amino acid residues. Polypeptides or nucleic acids ofthe disclosure may contain one or more conservative substitution.

As used throughout the disclosure, the term “more than one” of theaforementioned amino acid substitutions refers to 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 or more of the recitedamino acid substitutions. The term “more than one” may refer to 2, 3, 4,or 5 of the recited amino acid substitutions.

Polypeptides and proteins of the disclosure, either their entiresequence, or any portion thereof, may be non-naturally occurring.Polypeptides and proteins of the disclosure may contain one or moremutations, substitutions, deletions, or insertions that do notnaturally-occur, rendering the entire amino acid sequence non-naturallyoccurring. Polypeptides and proteins of the disclosure may contain oneor more duplicated, inverted or repeated sequences, the resultantsequence of which does not naturally-occur, rendering the entire aminoacid sequence non-naturally occurring. Polypeptides and proteins of thedisclosure may contain modified, artificial, or synthetic amino acidsthat do not naturally-occur, rendering the entire amino acid sequencenon-naturally occurring.

As used throughout the disclosure, “sequence identity” may be determinedby using the stand-alone executable BLAST engine program for blastingtwo sequences (bl2seq), which can be retrieved from the National Centerfor Biotechnology Information (NCBI) ftp site, using the defaultparameters (Tatusova and Madden, FEMS Microbiol Lett., 1999, 174,247-250; which is incorporated herein by reference in its entirety). Theterms “identical” or “identity” when used in the context of two or morenucleic acids or polypeptide sequences, refer to a specified percentageof residues that are the same over a specified region of each of thesequences. The percentage can be calculated by optimally aligning thetwo sequences, comparing the two sequences over the specified region,determining the number of positions at which the identical residueoccurs in both sequences to yield the number of matched positions,dividing the number of matched positions by the total number ofpositions in the specified region, and multiplying the result by 100 toyield the percentage of sequence identity. In cases where the twosequences are of different lengths or the alignment produces one or morestaggered ends and the specified region of comparison includes only asingle sequence, the residues of single sequence are included in thedenominator but not the numerator of the calculation. When comparing DNAand RNA, thymine (T) and uracil (U) can be considered equivalent.Identity can be performed manually or by using a computer sequencealgorithm such as BLAST or BLAST 2.0.

EXAMPLES Example 1: Production of Stem-Like Modified T-Cells

The following is an illustrative but nonlimiting example of one protocolfor modifying T cells to express a chimeric antigen receptor (CAR) underconditions that induce or preserve desirable stem-like properties of theT cells.

Day 0: Nucleofection of T Cells

Pre-warm ImmunoCult™-XF T cell expansion medium (Stemcell Technologies,Cat #: 10981) in 37° C., 5% CO2, high humidity incubator. For 5×10⁶ Tcells/reaction (100 μL cuvette size) warm 3 mL of media/reaction in asingle well of a 6-well plate. For 25×10⁶ T cells/reaction (100 μLcuvette size) warm 20 mL of media/reaction in a G-Rex10 (Wilson Wolf,Cat #: 80040S).

Warm P3 primary cell solution (Lonza, Cat #: PBP3-02250) up to roomtemperature and add supplement if necessary.

Turn on the core unit (Lonza, Cat #: AAF-1002B) of the 4D-Nucleofector™System, which controls the X-unit (Lonza, Cat #: AAF-1002X). Program thenumber of nucleofections required to use P3 buffer. Program EO-210.

Label cuvettes, pre-open transfer pipettes (supplied with the Lonza P3kit), and prepare proper dilutions of nucleic acids prior to workingwith the cells.

For a transposon plasmid, make a 0.5 μg/μL solution in nuclease freeH₂O.

Count CD14, CD56, and CD19 depleted cells collected using the CliniMACsProdigy and calculate the volume needed for the required cell number.

Centrifuge T cells at 90 g for 10 minutes with brake at 7 on a HeraeusMultifuge X3R benchtop centrifuge (Thermofisher Scientific). Ifperforming multiple reactions using the same number of cells/reaction,centrifuge all the necessary cells in a single centrifuge tube. Either a15 mL (Fisher, Cat #: 14-959-49B) or 50 mL (Fisher, Cat #: 14-959-49A)conical tube can be used depending on volume. During centrifugation addnucleic acids directly to the bottom of cuvettes that come with the P3primary cell solution box (Lonza, Cat #: PBP3-02250). Add 2 μL of the0.5 μg/μL transposon plasmid solution made in step 4 for a total of 1 μgtransposon to one of the bottom corners of the cuvette. Add 5 μg ofSuper piggyBac™ (SPB) transposase mRNA to the other corner of thecuvette.

Because mRNA can be rapidly degraded, it is optimal to minimize the timeit is in contact with other nucleic acid solutions and with cells priorto electroporation due to the potential presence of RNases. This is why,for example, the transposon and transposase are delivered to oppositecorners of the cuvette to prevent mixing. In addition, it is optimal tokeep the total volume of nucleic acids under 10 μL (10%) of the totalreaction volume.

The amount of both transposon (1 μg) and transposase (5 μg) stays thesame regardless of the number of cells/reaction. Transpositionefficiencies remain unchanged between 5×10⁶ cells/100 μL reaction and25×10⁶ cells/100 μL reaction.

Following centrifugation, completely aspirate off the media withoutdisturbing the cell pellet.

Suspend the cell pellet in 100 μL of room temperature P3 buffercontaining the supplement/reaction.

Transfer 100 μL of cells in P3 buffer to a cuvette containing theappropriate nucleic acids, optimally, taking care not to introduce anyair bubbles into the solution. It is recommended that only up to 2cuvettes should loaded with cells at a time. After the addition of cellsto the cuvette, it is optimal to work quickly and efficiently to reducecontact time of mRNA with cells prior to nucleofection. While nodecrease in transposition efficiency has observed for cells resting inP3 buffer for up to 10 minutes, it is recommended to minimize the amountof time cells remain in P3.

Mix the contents of the cuvette by flicking several times and load up totwo cuvettes into the 4D-Nucleofector™ X-unit.

Pulse the cells with program EO-210 and ensure there was no errorrecorded by the machine.

Immediately transfer the nucleofected cells into either the 6-well plateor G-Rex10 using the transfer pipettes provided with the Lonza P3 kit.To transfer the cells, first draw up a small amount of pre-warmed mediainto the transfer pipette from either the 6-well plate or the G-Rexflask. Then pipette the media into the cuvette and transfer the entirecontents of the cuvette using the pipette into the final culture dish.It is recommended not to pipette the cells up and down in either thecuvette or the final culture dish.

Repeat protocol from the transfer of cells in P3 buffer to a cuvettecontaining the appropriate nucleic acids through the mixing, pulsing,and transfer of the nucleofected cells into either the 6-well plate orG-Rex10 for any remaining reactions.

Place cells in incubator at 37° C., 5% CO2, high humidity.

Day 2: T Cell Activation

Add 25 μL/mL of ImmunoCult™ Human CD3/CD28/CD2 T cell Activator(Stemcell Technologies, Cat #: 10970) to the nucleofected cells.

Mix cells gently by pipetting.

Place cells back into the incubator at 37° C., 5% CO2, high humidity.

For Cells being Grown in G-Rex Flask:

It is essential not to disturb the cultures until visible cell clumpingis observed. Thus, it is recommended to separate the media additions andchanges from the disruption/mixing/pipetting of the cells.

Culture Media Notes:

For growing cells in the G-Rex flask, media addition and/or changesshould be done based off of glucose and other metabolite levels. If theglucose level (or another indicating metabolite) falls to a criticallevel (˜100 mg/dL of glucose, for example) media volume should bedoubled and/or replenished by a half-media change using pre-warmedImmunoCult™-XF T cell expansion medium. Media addition should beperformed slowly and care taken to disrupt the cells as little aspossible. Half media changes should be performed at least 12 hours postmechanical disruption of the cell culture to allow the cells to fullysettle to the bottom of the culture flask.

Cell Sampling and Disruption:

Cells should be left undisturbed during much of the culture period.

The first disruption of the cell culture following activation reagentaddition should occur once large visible aggregates of cells have formed(aggregates will measure 3-4 squares by 3-4 squares of the grid that canbe seen on the G-Rex membrane).

Once cell aggregates have reached the required size, they can bemechanically disrupted using a 10 mL serological pipette. This timepoint may occur between 11-14 days depending on donor and transpositionefficiency. In certain circumstances, this time point may occur closerto day 14 than day 11, for example, when using a manual cassette, alarge volume and/or a large cell number for nucleofection. A sampling ofcells should be collected at this point for cell counts, viability, andflow analysis. Ideally the volume of culture medium at this point willhave no more than doubled from the initial volume used (200 mL for aG-Rex100). It is recommended to collect all of the cells needed at onceso that the cells do not need to be disturbed again.

Once the cells have been disrupted they should be left undisturbed for12 hours in the same volume of media they started in. Cells shouldre-aggregate at this point; however, the aggregates will be smaller andmore numerous. These aggregates should measure 1-2 squares by 1-2squares on the G-Rex membrane grid.

Three days following the first disruption (day 14-17 depending on theculture) of the cells they can be pipetted a second time. Samples shouldbe taken again for cell counts, viability, and flow cytometry. Onceagain the cells should be left undisturbed for at least 12 hours postsampling. It is recommended to collect all of the cells needed at onceso that the cells do not need to be disturbed again.

Following this second disruption, the cells will likely not form anyclumps and the rate of cell growth will slow considerably.

Cell harvest should be performed 3 days after the second disruption ofcells between day 17 and day 20 of the culture.

Flow Cytometry

Flow should be run on Day 5, D-Day, D-Day+3, and D-Day+6.

For Day 5, D-Day, and D-Day+3 use the CD45, CD4, CD8, and CARTyrin flowpanel

For D-Day+6, there are 3 target panels:

a. Panel 1: CD3, CD8, CD4, CARTyrin, CD45RA, CD45RO, CD62L

b. Panel 2: CD3, CD8, CD4, CARTyrin, CD25, CXCR4, PD-1

c. Panel 3: CD45, CD14, CD20, CD56, CD8, CD4, CD3

Example 2: Functional Characterization of CARTyrin+ Stem Memory T Cells

CARTyrins of the disclosure may be introduced to T cells using a plasmidDNA transposon encoding the CARTyrin that is flanked by twocis-regulatory insulator elements to help stabilize CARTyrin expressionby blocking improper gene activation or silencing.

In certain embodiments of the methods of the disclosure, the piggyBac™(PB) Transposon System may be used for stable integration ofantigen-specific (including cancer antigen-specific) CARTyrin intoresting pan T cells, whereby the transposon was co-delivered along withan mRNA transposase enzyme, called Super piggyBac™ (SPB), in a singleelectroporation reaction. Delivery of piggyBac™ transposon intountouched, resting primary human pan T cells resulted in 20-30% of cellswith stable integration and expression of PB-delivered genes.Unexpectedly, a majority of these modified CARTyrin-expressing T cellswere positive for expression of CD62L and CD45RA, markers commonlyassociated with stem memory T-cells (T_(SCM) cells). To confirm thatthis phenotype was retained upon CAR-T cell stimulation and expansion,the modified CARTyrin-expressing T cells positive for expression ofCD62L and CD45RA were activated via stimulation of CD3 and CD28. As aresult of stimulation of CD3 and CD28, >60% of CARTyrin+ T cellsexhibited a stem-cell memory phenotype. Furthermore, these cells, whichexpressed a CARTyrin specific for a cancer antigen, were fully capableof expressing potent anti-tumor effector function.

To determine whether or not the PB system directly contributed toenhancing the expression of stem-like markers, the phenotype of CAR-Tcells generated either by PB transposition or lentiviral (LV)transduction was compared. To do this, a new vector was constructed bysubcloning the CARTyrin transgene into a common LV construct forproduction of virus. Following introduction of the CARTyrin to untouchedresting T cells either by PB-transposition or LV-transduction, theCARTyrin⁺ cells were expanded and then allowed to return to a restingstate. A variety of phenotypic and functional characteristics weremeasured including kinetic analysis of memory and exhaustion-associatedmarkers, secondary proliferation in response to homeostatic cytokine ortumor-associated Ag, cytokine production, and lytic capability inresponse to target tumor cells. Unlike the PB-transposed CARTyrin⁺ Tcells, the LV-transduced CARTyrin⁺ T cells did not exhibit an augmentedmemory phenotype. In addition, PB-transposed cells exhibited acomparable or greater capability for secondary proliferation and killingof target tumor cells. Together, these data demonstrate that CAR-T cellsproduced by PB transposition are predominantly T_(SCM) cells, a highlydesirable product phenotype in the CAR-T field. Furthermore, theseCARTyrin⁺ T cells exhibit strong anti-tumor activity and may give riseto cells that persist longer in vivo due to the use of a Centyrin-basedCAR, which may be less prone to tonic signaling and functionalexhaustion.

Example 3: Sleeping Beauty Transposition Yields Predominantly T_(SCM)Phenotype

Sleeping Beauty (SB100x) Transposition yielded a predominately T_(SCM)phenotype using the methods of the disclosure. Human pan T cells weretransposed using 1 μg of either a Sleeping Beauty or piggyBac transposonplasmid and SB100x or SPB mRNA, respectively as shown in FIG. 10.Following transposition, cells were expanded ex vivo and allnon-transposed cells were depleted using a drug selection system.Following 18 days in culture, cells were stained with the phenotypicmarkers CD4, CD8, CD45RA, and CD62L. Stem cell memory phenotype(T_(SCM)) is defined by CD45RA and CD62L double positive cells and makeup >65% of the cells in all of samples.

Example 4: Expression of Factor IX in Modified T-Cells

Genetic deficiencies in Factor IX (FIG. 11) lead to a life threateningdisease called Hemophila B. Hemophila B is a rare disease that affects 1in 25,000 to 1 in 30,000 people. Current Hemophilia B treatments involvean infusion of recombinant Factor IX protein every 2-3 days, at a costof around $250,000 per year.

Stem memory T cells (T_(SCM) cells) are maintained in humans for severaldecades, and are therefore an ideal vehicle to secrete Factor IX,supplying the Factor IX missing in Hemophilia B patients without theneed for frequent transfusions. T cells were transformed with PiggyBacto secrete Factor IX. When transgenic T cells encoding a human Factor IXtransgene were examined for T and T_(SCM) cell markers using FACS,approximately 80% of all cells showed a T_(SCM) phenotype (FIG. 12).These modified T cells were able to secrete human Factor IX (FIG. 13A),and this secreted Factor IX provided clotting activity (FIG. 13B).

INCORPORATION BY REFERENCE

Every document cited herein, including any cross referenced or relatedpatent or application is hereby incorporated herein by reference in itsentirety unless expressly excluded or otherwise limited. The citation ofany document is not an admission that it is prior art with respect toany invention disclosed or claimed herein or that it alone, or in anycombination with any other reference or references, teaches, suggests ordiscloses any such invention. Further, to the extent that any meaning ordefinition of a term in this document conflicts with any meaning ordefinition of the same term in a document incorporated by reference, themeaning or definition assigned to that term in this document shallgovern.

OTHER EMBODIMENTS

While particular embodiments of the disclosure have been illustrated anddescribed, various other changes and modifications can be made withoutdeparting from the spirit and scope of the disclosure. The scope of theappended claims includes all such changes and modifications that arewithin the scope of this disclosure.

What is claimed is:
 1. A method of producing a plurality of expandedmodified T cells, comprising introducing into a plurality of primaryhuman T cells (a) a transposon composition comprising a transposoncomprising an antigen receptor, a therapeutic protein or a sequenceencoding the same and (b) a transposase composition comprising atransposase or a sequence encoding the transposase to produce aplurality of modified T cells, under conditions that preserve desirablestem-like properties of the plurality of modified T cells; wherein theconditions comprise culturing the plurality of modified T cells in vitroin a liquid culture media comprising a sterol; an alkane; phosphorus andone or more of an octanoic acid, a palmitic acid, a linoleic acid, andan oleic acid; wherein at least 25% of the plurality of modified T cellsexpresses one or more cell-surface marker(s) of a stem memory T cell(T_(SCM)) or a T_(SCM)-like cell and wherein the one or morecell-surface marker(s) comprises CD62L and CD45RA.
 2. The method ofclaim 1, wherein at least 60% of the plurality of modified T cellsexpresses one or more cell-surface marker(s) of a stem memory T cell(T_(SCM)) or a T_(SCM)-like cell.
 3. The method of claim 1, wherein thetransposon is a plasmid DNA transposon with a sequence encoding theantigen receptor or the therapeutic protein flanked by twocis-regulatory insulator elements.
 4. The method of claim 1, wherein thetransposon is a piggyBac transposon.
 5. The method of claim 4, whereinthe transposase is a piggyBac transposase.
 6. The method of claim 5,wherein the piggyBac transposase comprises an amino acid sequencecomprising SEQ ID NO:
 4. 7. The method of claim 6, wherein the piggyBactransposase is a hyperactive variant and wherein the hyperactive variantcomprises an amino acid substitution at one or more of positions 30,165, 282 and 538 of SEQ ID NO:
 4. 8. The method of claim 7, wherein theamino acid substitution at position 30 of SEQ ID NO: 4 is a substitutionof a valine (V) for an isoleucine (I) (I30V).
 9. The method of claim 7,wherein the amino acid substitution at position 165 of SEQ ID NO: 4 is asubstitution of a serine (S) for a glycine (G) (G165S).
 10. The methodof claim 7, wherein the amino acid substitution at position 282 of SEQID NO: 4 is a substitution of a valine (V) for a methionine (M) (M282V).11. The method of claim 7, wherein the amino acid substitution atposition 538 of SEQ ID NO: 4 is a substitution of a lysine (K) for anasparagine (N) (N538K).
 12. The method of claim 4, wherein thetransposase is a Super piggyBac (SPB) transposase.
 13. The method ofclaim 12, wherein the Super piggyBac (SPB) transposase comprises anamino acid sequence comprising SEQ ID NO:
 5. 14. The method of claim 1,wherein the sequence encoding the transposase is an mRNA sequence. 15.The method of claim 1, wherein the transposon is a Sleeping Beautytransposon.
 16. The method of claim 15, wherein the transposase is aSleeping Beauty transposase or a hyperactive Sleeping Beauty transposase(SB100X).
 17. The method of claim 1, wherein the transposon is aHelraiser transposon.
 18. The method of claim 17, wherein thetransposase is a Helitron transposase.
 19. The method of claim 1,wherein the transposon is a Tol2 transposon.
 20. The method of claim 19,wherein the transposase is a Tol2 transposase.
 21. The method of claim1, wherein the transposon is derived or recombined from any species. 22.The method of claim 1, wherein the transposon is synthetic.
 23. Themethod of claim 1, wherein the antigen receptor is a T-cell receptor.24. The method of claim 23, wherein the T-cell receptor is notnaturally-occurring.
 25. The method of claim 1, wherein the antigenreceptor is a Chimeric Antigen Receptor (CAR).
 26. The method of claim25, wherein the CAR is a CARTyrin.
 27. The method of claim 1, whereinthe therapeutic protein is a secreted or secretable protein.
 28. Themethod of claim 25, wherein the CAR is a VCAR.